DETECTION AND USE OF ANTIVIRAL RESISTANCE MUTATIONS

Abstract

The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. Vaccines and diagnostic assays are also contemplated herein.

Description

[0001] This application is a continuation under 35 U.S.C. 120 of U.S. application Ser. No. 12/303,942, filed Jun. 4, 2007, now U.S. Pat. No. 8,859,198, which was the U.S. National Phase of International Application PCT/AU2007/000785, filed Jun. 4, 2007 designating the U.S., and published in English as WO 2007/140522 on Dec. 13, 2007, which claims priority to Australian Patent Application No. 2006903065 filed Jun. 6, 2006.

FIELD

[0002] The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. Vaccines and diagnostic assays are also contemplated herein.

BACKGROUND

[0003] Bibliographic details of the publications referred to in this specification are also collected at the end of the description.

[0004] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country.

[0005] Hepatitis B virus (HBV) can cause debilitating disease conditions and can lead to acute liver failure. HBV is a DNA virus which replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy (Summers and Mason, Cell 29:403-415, 1982). The HBV genome is of a complex nature having a partially double-stranded DNA structure with overlapping open reading frames encoding surface, core, polymerase and X genes. The complex nature of the HBV genome is represented in FIG. 1. The polymerase consists of four functional regions, the terminal protein (TP), spacer, reverse transcriptase (rt) and ribonuclease (RNAse).

[0006] The polymerase gene of HBV overlaps the envelope gene, mutations in the catalytic domain of the polymerase gene can also affect the nucleotide and the deduced amino acid sequence of the envelope protein and vice versa. In particular, the genetic sequence for the neutralization domain of HBV known as the `a` determinant, which is found within the HBsAg and located between amino acids 99 and 169, actually overlaps the major catalytic regions of the viral polymerase protein and in particular domains A and B.

[0007] The presence of an HBV DNA polymerase has led to the proposition that nucleoside or nucleotide analogs could act as effective anti-viral agents. Examples of nucleoside or nucleotide analogs currently being tested are penciclovir and its oral form (FCV) [Vere Hodge, Antiviral Chem Chemother 4: 67-84, 1993; Boyd et al, Antiviral Chem Chemother. 32:358-363, 1987; Kruger et al, Hepatology 22:219 A, 1994; Main et al, J. Viral Hepatitis 3:211-215, 1996], Lamivudine [(-)-β-2'-deoxy-3'-thiacytidine]; (3TC or LMV) [Severin et al, Antimicrobial Agents Chemother. 39:430-435, 1995; Dienstag et al, New England J Med 333:1657-1661, 1995]. New nucleoside or nucleotide analogs which have already progressed to clinical trials include the pyrimidines Emtricitabine, ((-)-β-L-2'-3'-dideoxy-5-fluoro-3'-thiacydidine; FTC), the 5-fluoro derivative of 3TC, and Clevudine (1-(2-fluoro-5-methyl-β-L-arabino-furanosyl) uracil; L-FMAU), a thymidine analog. The beta-Lthymidine analogue (LdT has recently been given FDA approval other similar compounds include beta-L-2'-deoxycytidine (LdC) and beta-L-2'-deoxyadenosine (LdA) [Standring et al., Antivir Chem Chemother. 2001; 12 Suppl 1:119-29]. Like 3TC, these are pyrimidine derivatives with an unnatural "L"--configuration. Several purine derivatives have also progressed to clinical trials; they include Entecavir (BMS-200, 475; ETV), a carbocyclic deoxyguanosine analog, diaminopurine dioxolane (DAPD), an oral pro-drug for dioxolane guanine ((-)-β-D-2-aminopurine dioxolane; DXG) and Adefovir dipivoxil, an oral prodrug for the acyclic deoxyadenosine monophosphate nucleoside or nucleotide analog Adefovir (9[phosphoryl-methoxyethyl]-adenine; PMEA). Other drugs in pre-clinical and clinical trials include FLG [Medivir], ACH-126,443 (L-d4C) [Archillion Pharmaceuticals], ICN 2001-3 (ICN) and Racivir (RCV) [Pharmassett].

[0008] Whilst these agents are highly effective in inhibiting HBV DNA synthesis, there is the potential for resistant mutants of HBV to emerge during long term antiviral chemotherapy. In patients on prolonged LMV therapy, key resistance mutations are selected in the rt domain within the polymerase at rtM204IN+/-rtL180M as well as other mutations. The nomenclature used for the polymerase mutations is in accordance with that proposed by Stuyver et al, 2001, supra. LMV is a nucleoside or anucleotide analog that has been approved for use against chronic HBV infection. LMV is a particularly potent inhibitor of HBV replication and reduces HBV DNA titres in the sera of chronically infected patients after orthotopic liver transplantation (OLT) by inhibiting viral DNA synthesis. LMV monotherapy seems unlikely to be able to control HBV replication in the longer term. This is because emergence of LMV-resistant strains of HBV seems almost inevitable during monotherapy.

[0009] Adefovir dipivoxil (ADV: formerly, bis-pom PMEA) is an orally available prodrug of the acyclic deoxyadenosine monophosphate analog adefovir (formerly, PMEA) (FIG. 2). ADV is also a potent inhibitor of HBV replication and has been given FDA approval for use against chronic HBV infection. Adefovir dipivoxil differs from other agents in this class in that it is a nucleotide (vs. nucleoside) analog and as such bypasses the first phosphorylation reaction during drug activation. This step is often rate-limiting. Adefovir dipivoxil has demonstrated clinical activity against both wild-type and lamivudine-resistant strains of HBV and is currently in phase III clinical Testing (Gilson et al, J Viral Hepat 6:387-395, 1999; Perrino et al, Hepatology 32:129-134, 2000; Peters et al, Transplantation 68:1912-1914, 1999; Benhamou et al, Lancet 358:718-723, 2001). During phase II studies a 30 mg daily dose of adefovir dipivoxil resulted in a mean 4 log₁₀ decrease in viremia over 12 weeks (Heathcote et al, Hepatology 28:A620, 1998).

[0010] ADV is a substituted acyclic nucleoside phosphonate. This class of compounds also includes tenofovir disoproxil fumarate (also referred to as tenofovir DF, or tenofovir, or (TFV) or 9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and is marketed as Viread by Gilead sciences).

[0011] TFV has antiviral activity against both HBV and HIV (Ying et al, J Viral Hepat. 7(2):161-165, 2000; Ying et al, J. Viral Hepat. 7(1):79-83, 2000, Suo et al, J Biol Chem. 273(42):27250-27258. 1998).

[0012] FTC has activity against HBV and HIV (Frick et al, Antimicrob Agents Chemother 37:2285-2292, 1993).

[0013] LdT, LdC and LdA have activity against HBV (Standring et al, in supra)

[0014] Nucleoside or nucleotide analog therapy may be administered as monotherapy or combination therapy where two or more nucleoside or nucleotide analogs may be administered. The nucleoside or nucleotide analogs may also be administered in combination with other antiviral agents such as interferon or hepatitis B immunoglobulin (HBIG).

[0015] There is a need to monitor for the emergence of nucleoside/nucleotide-analog- or antibody-resistant strains of HBV and to develop diagnostic protocols to detect these resistant viruses and/or to use them to screen for and/or develop or design agents having properties making them useful as anti-viral agents. Defective forms of these resistant strains or antigenic components therefrom are also proposed to be useful in the development of therapeutic vaccine compositions as are antibodies directed to viral surface components.

SUMMARY

[0016] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0017] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

[0018] Specific mutations in an amino acid sequence are represented herein as "Xaa₁nXaa₂" where Xaa_i is the original amino acid residue before mutation, n is the residue number and Xaa₂ is the mutant amino acid. The abbreviation "Xaa" may be the three letter or single letter (i.e. "X") code. An "rt" before " Xaa₁nXaa₂" means "reverse transcriptase". An "s" means an envelope gene. The amino acid residues for HBV DNA polymerase are numbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD) being residue number 204 (Stuyver et al, Hepatology 33:751-757, 2001). The amino acid residues for hepatitis B virus surface antigen are number according to Norder et al, (J. Gen. Virol. 74:341-1348, 1993). Both single and three letter abbreviations are used to define amino acid residues and these are summarized in Table 2.

[0019] The selection of HBV variants is identified in patients with chronic HBV infection treated with antiviral agents including nucleosides and nucleotide analogs including TFV and/or LMV, or LMV and/or ADV and/or ETV. Consequently, HBV rt variants are contemplated which are resistant to, or which exhibit reduced sensitivity to antiviral agents including nucleosides and nucleotide analogs including, ADV, LMV, TFV, ETV or FTC, or LdT; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof. Corresponding mutations in the surface antigen also occur. The identification of these HBV variants is important for the development of assays to monitor ADV, LMV, FTC, TFV, LdT and/or ETV resistance and/or resistance to other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and to screen for agents which are useful as alternative therapeutic agents.

[0020] Reference herein to "anti-HBV agents" includes nucleoside and nucleotide analogs as well as immunological reagents (e.g. antibodies to HBV surface components) and chemical, proteinaceous and nucleic acid agents which inhibit or otherwise interfere with viral replication, maintenance, infection, assembly or release.

[0021] The detection of such HBV variants is particularly important in the management of therapeutic protocols including the selection of appropriate agents for treating HBV infection. The method of this aspect of the present invention is predicated in part on monitoring the development in a subject of an increased HBV load in the presence of a nucleoside or nucleotide analog or other anti-HBV agents or combinations thereof. The clinician is then able to modify an existing treatment protocol or select an appropriate treatment protocol accordingly.

[0022] Accordingly, one aspectis directed to an isolated Hepatitis B virus (HBV) variant wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase and wherein said variant exhibits decreased sensitivity to one or more nucleoside or nucleotide analogs selected from the list consisting of ADV, LMV, TFV, ETV, LdT or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof. The variant HBV comprises a mutation in an overlapping open reading frame in its genome in a region defined by one or more of domains F and G and domain A through to E of HBV DNA polymerase.

[0023] Another aspect provides an isolated HBV variant comprising a nucleotide mutation in the S gene resulting in at least one amino acid addition, substitution and/or deletion to the surface antigen and which exhibits decreased sensitivity to one or more nucleoside or nucleotide analogs selected from the list consisting of ADV, LMV, TFV, ETV, or LdT or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof:

[0024] Useful mutants in the rt region include, in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation.

[0025] Particularly useful mutants are co-mutations at codons 180, 184 and 204 such as rtL180M and rtT184S and rtM204V, or co-mutations at codons 85, 180, 184, 202, 204 and 236 such as rtS85T, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN. Alternative useful mutations contemplated herein include rtP170H, rtT184A, rtY221H/Y, rtI233T/I, rtT240N/T, rtK241R, rtR242K/R, rtW243R/W, rtI253V, rtI254I/F, rtS256R/S rtW257G/W, rtY257H or mutations at codon 239.

[0026] Mutations in the putative tri-phosphate binding site at rtN236T region are proposed herein to be important in antiviral resistance against ADV and/or TFV and/or other nucleoside or nucleotide treatments. Important codons apart from codons 236 include codons 239 which is highly conserved and codons 240, 241, 242 and 243.

[0027] In a particular embodiment, an isolated Hepatitis B virus (HBV) variant is provided wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase wherein the mutation is selected from the group consisting of: (i) a mutation at codon 239, 240, 241, 242 and/or 243; (ii) a mutation resulting in an N236I/DN substitution; (iii) a mutation resulting in an S246H/P/Y/S substitution; (iv) a mutation resulting in a P170H substitution; (v) a mutation resulting in an I253V substitution; (vi) co-mutations of two or more substitutions selected from the list consisting of rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN; (vii) co-mutations of two or more substitutions selected from the list consisting of rtF61L, rtS78T, rtV84MN, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S; and (viii) co-mutations of two or more substitutions selected from the list consisting of rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V; wherein the HBV variant exhibits decreased sensitivity to a nucleoside or nucleotide analog.

[0028] Useful mutations in the S gene include, in one embodiment include sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL162I and sI195M or a combination thereof or an equivalent mutation.

[0029] Particularly useful mutants are sI110V, sP120Q, sF134L/F, sL162I, sL173F, sL175F, sL176V and sP178P/L.

[0030] A method is further contemplated for determining the potential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof by isolating DNA or corresponding mRNA from the HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and G and domains A through to E or a region proximal thereto of the DNA polymerase and associated with resistance or decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0031] The presence of such a mutation is an indication of the likelihood of resistance to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0032] Also provided is a composition comprising a variant HBV resistant to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof or an HBV surface antigen from the variant HBV or a recombinant or derivative form thereof or its chemical equivalent and one or more pharmaceutically acceptable carriers and/or diluents.

[0033] Yet another aspect provides a use of the aforementioned composition or a variant HBV comprising a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to the DNA polymerase and a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof in the manufacture of a medicament for the treatment and/or prophylaxis of hepatitis B virus infection.

[0034] Also contemplated is a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other anti-HBV agents or by isolating DNA or corresponding mRNA from the HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase wherein the presence of the following mutations in the rt region: in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and Thy; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0035] Still a further methodology comprises screening for a mutation in the nucleotide sequence encoding the envelope genes (s) wherein the presence of the following mutations in the s gene: in one embodiment include sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment include sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL162I and sI195M or combinations thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof, and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof.

[0036] In a particular embodiment, the variants are in an isolated form such that they have undergone at least one purification step away from naturally occurring body fluid. Alternatively, the variants may be maintained in isolated body fluid or may be in DNA form. The present invention also contemplates infectious molecular clones comprising the genome or parts thereof from a variant HBV. The detection of HBV or its components in cells, cell lysates, cultured supernatant fluid and bodily fluid may be by any convenient means including any nucleic acid-based detection means, for example, by nucleic acid hybridization techniques or via one or more polymerase chain reactions (PCRs). The term "bodily fluid" includes any fluid derived from the blood, lymph, tissue or organ systems including serum, whole blood, biopsy and biopsy fluid, organ explants and organ suspension such as liver suspensions.

[0037] Another aspect is directed to a variant HBV comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to a surface antigen from a reference or wild type HBV and wherein an antibody generated to the reference or wild type surface antigen exhibits an altered immunological profile relative to the HBV variant. One altered profile includes a reduced capacity for neutralizing the HBV. More particularly, the surface antigen of the variant HBV exhibits an altered immunological profile compared to a pre-treatment HBV where the variant HBV is selected for by a nucleoside or nucleotide analog or other anti-HBV agents of the HBV DNA polymerase. The variant HBV of this aspect of the invention may also comprise a nucleotide sequence comprising a single or multiple nucleotide substitution, addition and/or deletion compared to a pre-treatment HBV.

[0038] Further contemplated is a method for detecting a variant HBV exhibiting an altered immunological profile said method comprising isolating an HBV from a subject exposed to a nucleoside or nucleotide analog or combination of analogs selected from the listed consisting of ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof, and then contacting said HBV with a panel of one or more antibodies to a surface antigen and screening for any change in binding affinity or binding spectrum.

[0039] In a related aspect, a method is provided for detecting a variant HBV exhibiting an altered immunological profile said method comprising isolating a serum sample from a subject exposed to a nucleoside or nucleotide analog selected from the listed consisting of ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof, and then contacting the serum with a panel of HBV surface antigens or antibody-binding fragments thereof and screening for any change in binding affinity or binding spectrum.

[0040] An isolated HBsAg is provided or a recombinant form thereof or derivative or chemical equivalent thereof corresponding to the variant HBV. Generally, the HBsAg or its recombinant or derivative form or its chemical equivalent comprises an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to an HBsAg from a reference HBV and wherein an antibody directed to a reference HBV exhibits an altered immunological profile to an HBV carrying said variant HBsAg. In one embodiment, the altered immunological profile comprises a reduction in the ability to neutralize the variant HBV.

[0041] Another aspect contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV by generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in a plasmid vector and then transfecting said cells with said construct, contacting the cells, before, during and/or after transfection, with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agents; and the subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent. In a preferred embodiment, the plasmid vector in a baculovirus vector and the method comprises generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct, contacting the cells, before, during and/or after infection, with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

[0042] In connection with these methods, the plasmid vector may include genes encoding part or all of other viral vectors such as baculovirus vectors or adenovirus vectors (see Ren and Nassal, J. Virol. 75(3):1104-1116, 2001).

[0043] In an alternative embodiment, the method comprises generating a continuous cell line comprising an infectious copy of the genome of the HBV in a replication competent effective amount such that said infectious HBV genome is stably integrated into said continuous cell line such as but not limited to the 2.2.15 or AD cell line, contacting the cells with the agent to be tested, culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to the agent and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

[0044] In an alternative embodiment, a method is contemplated for detecting an agent which exhibits inhibitory activity to an HBV polymerase in an in vitro polymerase assay. The HBV polymerase activity can be examined using established assays (Gaillard et al, Antimicrob Agents Chemother. 46(4):1005-1013, 2002; Xiong et al, Hepatology, 28(6):1669-73, 1998). The HBV polymerase may be a wild-type or reference HBV polymerase or mutant HBV polymerase.

[0045] The identification of viral variants enables the production of vaccines comprising particular recombinant viral components such as polymerases or envelope genes PreS1, PreS2, S encoding for L, M, S proteins as well as therapeutic vaccines comprising defective HBV variants. Rational drug design may also be employed to identify or generate therapeutic molecules capable of interacting with a polymerase or envelope genes PreS1, PreS2, S encoding for L, M, S proteins or other component of the HBV. Such drugs may also have diagnostic potential. In addition, defective HBV variants may also be used as therapeutic compositions to generate an immune response against the same, similar or homologous viruses. Alternatively, antibodies generated to the HBV variants or surface components thereof may be used in passive immunization of subjects against infection by HBV variants or similar or homologous viruses. Furthermore, agents such as nucleoside or nucleotide analogs, RNAi or siRNA molecules (both DNA-derived or synthetic), antisense or sense oligonucleotides, chemical or proteinaceous molecules having an ability to down-regulate the activity of a component of HBV and inhibit replication, maintenance, infection, assembly or release are contemplated by the present invention.

[0046] A summary of the abbreviations used throughout the subject specification are provided in Table 3.

[0047] A summary of sequence identifiers used throughout the subject specification is provided in Table 1.

TABLE-US-00001 TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1 PCR primer OSI 5' 2 PCR primer TTA3 5' 3 PCR primer JM 5' 4 PCR primer TTA4 5' 5 PCR primer OS2 5' 6 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient A 7 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient A 8 Deduced amino acid sequence of envelope gene from resistant HBV from Patient A 9 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient B 10 Deduced amino acid sequence of envelope gene from resistant HBV from Patient B 11 Deduced amino acid sequence of envelope gene from resistant HBV from Patient B 12 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient C 13 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient C 14 Deduced amino acid sequence of envelope gene from resistant HBV from Patient C 15 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient D 16 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient D 17 Deduced amino acid sequence of envelope gene from resistant HBV from Patient D 18 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient E 19 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient E 20 Deduced amino acid sequence of envelope gene from resistant HBV from Patient E 21 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient F 22 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient F 23 Deduced amino acid sequence of envelope gene from resistant HBV from Patient F 24 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient G 25 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient G 26 Deduced amino acid sequence of envelope gene from resistant HBV from Patient G 27 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient H 28 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient H 29 Deduced amino acid sequence of envelope gene from resistant HBV from Patient H 30 Nucleotide sequence of catalytic region of polymerase from resistant HBV Patient I 31 Deduced amino acid sequence of catalytic region of polymerase resistant HBV Patient I 32 Deduced amino acid sequence of envelope gene from resistant HBV from Patient I

TABLE-US-00002 TABLE 2 Single and three letter amino acid abbreviations Amino Acid Three-letter Abbreviation One-letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine The T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

TABLE-US-00003 TABLE 3 Abbreviations ABBREVIATION DESCRIPTION 3TC (LMV); (-)-β-2'-deoxy-3'-thiacytidine ADV adefovir dipivoxil DAPD diaminopurine dioxalone DXG dioxolane guanine ETV entecavir FAM famciclovir FCV famciclovir FTC emtricitabine HBIG hepatitis B immunoglobulin HBsAg hepatitis B surface antigen HBV hepatitis B virus LdT beta-Lthymidine LMV lamividuine PMEA 9-[phosphonyl-methoxyethyl]-adenine; adefovir PMPA 9-R-(2-phosphonomethoxypropyl)adenine RNase ribonuclease rt ("rt" before "Xaa₁nXaa₂" reverse transcriptase means reverse transcriptase) s (as used in a mutation, envelope gene e.g. sF134V) TFV tenofovir disoproxil fumarate YMDD Tyr Met Asp Asp-a motif in the polymerase protein; where the Met residue is designated residue number 204 of the reverse transcriptase

BRIEF DESCRIPTION OF THE FIGURES

[0048] FIG. 1 is a diagrammatic representation showing the partially double stranded DNA HBV genome showing the overlapping open reading frames encoding surface (S), core (C), polymerase (P) and X gene.

[0049] FIG. 2 is a diagrammatic representation of the chemical structure of ADV. FIG. 3 is a diagrammatic representation of the chemical structure of Tenofovir.

[0050] FIG. 4 is a representation showing comparison of the HBV nucleotide sequence (SEQ ID NO: 6) encoding the catalytic region of the polymerase gene in samples from Patient A.

[0051] FIG. 5 is a representation showing comparison of the deduced amino acid sequence SEQ ID NO: 7 of the catalytic region of the polymerase gene in samples from Patient A.

[0052] FIG. 6 is a representation showing comparison of the deduced amino acid sequence SEQ ID NO: 8 of the envelope gene in samples from Patient A.

[0053] FIGS. 7A and 7B are representations showing a comparison of the HBV nucleotide sequence encoding the catalytic region of the polymerase gene in samples from Patient B.

[0054] FIG. 8 is a representation showing comparison of the deduced amino acid sequence SEQ ID NO: 10 of the catalytic region of the polymerase gene in samples from Patient B

[0055] FIG. 9 is a representation showing comparison of the deduced amino acid sequence SEQ ID NO: 11 of the envelope gene in samples from Patient B.

[0056] FIG. 10 is a representation the HBV nucleotide sequence SEQ ID NO: 12 that also includes the encoding the catalytic region of the polymerase gene in samples from Patient C

[0057] FIG. 11 is a representation the deduced amino acid sequence SEQ ID NO: 13 of the polymerase gene in samples from Patient C.

[0058] FIG. 12 is a representation the deduced amino acid sequence SEQ ID NO: 14 of the envelope gene in samples from Patient C.

[0059] FIG. 13 is a representation the HBV nucleotide sequence SEQ ID NO: 15 encoding the catalytic region of the polymerase gene in samples from Patient D.

[0060] FIG. 14 is a representation the deduced amino acid sequence SEQ ID NO: 16 of the catalytic region of the polymerase gene in samples from Patient D.

[0061] FIG. 15 is a representation the deduced amino acid sequence SEQ ID NO: 17 of the envelope gene in samples from Patient D.

[0062] FIG. 16 is a representation the HBV nucleotide sequence SEQ ID NO: 18 encoding the catalytic region of the polymerase gene in samples from Patient E.

[0063] FIG. 17 is a representation the deduced amino acid sequence SEQ ID NO: 19 of the catalytic region of the polymerase gene in samples from Patient E.

[0064] FIG. 18 is a representation the deduced amino acid sequence SEQ ID NO: 20 of the envelope gene in samples from Patient E.

[0065] FIG. 19 is a representation the HBV nucleotide sequence SEQ ID NO: 21 encoding the catalytic region of the polymerase gene in samples from Patient F.

[0066] FIG. 20 is a representation the deduced amino acid sequence SEQ ID NO: 22 of the catalytic region of the polymerase gene in samples from Patient F.

[0067] FIG. 21 is a representation the deduced amino acid sequence SEQ ID NO: 23 of the envelope gene in samples from Patient F.

[0068] FIG. 22 is a representation the HBV nucleotide sequence SEQ ID NO: 24 encoding the catalytic region of the polymerase gene in samples from Patient G.

[0069] FIG. 23 is a representation the deduced amino acid sequence SEQ ID NO: 25 of the catalytic region of the polymerase gene in samples from Patient G.

[0070] FIG. 24 is a representation the deduced amino acid sequence SEQ ID NO: 26 of the envelope gene in samples from Patient G.

[0071] FIG. 25 is a representation the HBV nucleotide sequence SEQ ID NO: 27 encoding the catalytic region of the polymerase gene in samples from Patient H.

[0072] FIG. 26 is a representation the deduced amino acid sequence SEQ ID NO: 28 of the catalytic region of the polymerase gene in samples from Patient H.

[0073] FIG. 27 is a representation the deduced amino acid sequence SEQ ID NO: 29 of the envelope gene in samples from Patient H.

[0074] FIG. 28 is a representation the HBV nucleotide sequence SEQ ID NO: 30 encoding the catalytic region of the polymerase gene in samples from Patient I.

[0075] FIG. 29 is a representation the deduced amino acid sequence SEQ ID NO: 31 of the catalytic region of the polymerase gene in samples from Patient I.

[0076] FIG. 30 is a representation the deduced amino acid sequence SEQ ID NO: 32 of the envelope gene in samples from Patient I.

DETAILED DESCRIPTION

[0077] The present invention is predicated in part on the identification and isolation of nucleoside or nucleotide analog-resistant variants of HBV following treatment of patients with either ADV or LMV or ETV or more particularly ADV and LMV or TFV and LMV, or ETV and optionally one or more other nucleoside analogs or nucleotide analogs or other anti-HBV agents such as TFV, LdT, or FTC. In particular, ADV or ADV and LMV or ETV treated patients gave rise to variants of HBV exhibiting decreased or reduced sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof. Reference herein to "decreased" or "reduced" in relation to sensitivity to ADV and/or LMV and/or FTC and/or TFV and/or ETV includes and encompasses a complete or substantial resistance to the nucleoside or nucleotide analog or other anti-HBV agents as well as partial resistance and includes a replication rate or replication efficiency which is more than a wild-type in the presence of a nucleoside or nucleotide analog or other anti-HBV agents. In one aspect, this is conveniently measured by an increase in viral load during treatment, or alternatively, there is no substantial decrease in HBV DNA viral load from pre-treatment HBV DNA levels during treatment (i.e., non-response to treatment).

[0078] Accordingly, one aspect contemplates an isolated Hepatitis B virus (HBV) variant wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase and wherein said variant exhibits decreased sensitivity to one or more nucleoside or nucleotide analogs selected from the list consisting of ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0079] Another aspect provides a method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside or nucleotide analog selected from ADV, LMV, TFV, LdT, FTC and ETV or a combination thereof or optionally other nucleoside or nucleotide analogs, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of said DNA polymerase and associated with resistance or decreases sensitivity to one or more of ADV, LMV, TFV, FTC and/or wherein the presence of such a mutation is an indication of the likelihood of resistance to said one or more of ADV, LMV, TFV, LdT, FTC and/or ETV.

[0080] A further aspect provides an isolated Hepatitis B virus (HBV) variant is provided wherein said variant comprises a nucleotide mutation in a gene encoding a DNA polymerase resulting in at least one amino acid addition, substitution and/or deletion to said DNA polymerase wherein the mutation is selected from the group consisting of: (i) a mutation at codon 239, 240, 241, 242 and/or 243; (ii) a mutation resulting in an N236I/DN substitution; (iii) a mutation resulting in an S246H/P/Y/S substitution; (iv) a mutation resulting in a P170H substitution; (v) a mutation resulting in an I253V substitution; (vi) co-mutations of two or more substitutions selected from the list consisting of rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS2021, rtM204V and rtN236I/DN; (vii) co-mutations of two or more substitutions selected from the list consisting of rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rt1233T/I and rtS246H/P/Y/S; and (viii) co-mutations of two or more substitutions selected from the list consisting of rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V; wherein the HBV variant exhibits decreased sensitivity to a nucleoside or nucleotide analog.

[0081] Unless otherwise indicated, the present disclosure is not limited to specific formulations of components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0082] It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleoside or nucleotide analog" includes a single analog, as well as two or more analogs; reference to "an HBV variant" includes reference to two or more HBV variants; reference to "the invention" includes reference to a single or multiple aspect of the invention; and so forth.

[0083] The terms "analog", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound that induces a desired effect such as inhibit viral replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "analog", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.

[0084] Therefore, compounds are contemplated which are useful in inhibiting HBV replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Reference to an "analog", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" such as ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof. A "combination" also includes a two-part or more such as a multi-part anti-HBV therapeutic composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.

[0085] The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the agent to provide the desired therapeutic or physiological effect of inhibiting HBV replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Furthermore, an "effective HBV-inhibiting amount" or "effective symptom-ameloriating amount" of an agent is a sufficient amount of the agent to directly or indirectly inhibit replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.

[0086] By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0087] Similarly, a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

[0088] The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence, of symptoms and/or their underlying cause, and improvement or remediation of damage in relation to HBV infection. Thus, for example, "treating" a patient involves prevention of HBV infection as well as treatment of a clinically HBV symptomatic individual by inhibiting HBV replication, infection, maintenance, assembly and/or the function of an enzyme such as HBV DNA polymerase. Thus, for example, the present method of "treating" a patient with HBV infection or with a propensity for one to develop encompasses both prevention of HBV infection as well as treating HBV infection or symptoms thereof. In any event, the present invention contemplates the treatment or prophylaxis of HBV infection.

[0089] "Patient" as used herein refers to an animal, preferably a mammal and more preferably a primate including a lower primate and even more preferably, a human who can benefit from the formulations and methods of the present invention. A patient regardless of whether a human or non-human animal may be referred to as an individual, subject, animal, host or recipient. The compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. For convenience, an "animal" includes an avian species such as a poultry bird (including ducks, chicken, turkeys and geese), an aviary bird or game bird. The condition in a non-human animal may not be a naturally occurring HBV infection but HBV-like infection may be induced.

[0090] As indicated above, the preferred animals are humans, non-human primates such as marmossets, baboons, orangatangs, lower primates such as tupia, livestock animals, laboratory test animals, companion animals or captive wild animals. A human is the most preferred target. However, non-human animal models may be used.

[0091] Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Non-mammalian animals such as avian species, zebrafish, amphibians (including cane toads) and Drosophila species such as Drosophila melanogaster are also contemplated. Instead of a live animal model, a test system may also comprise a tissue culture system.

[0092] An "anti-HBV agent" includes a nucleoside or nucleotide analog, protein, chemical compound, RNA or DNA or RNAi or siRNA oligonucleotide (either DNA-derived or synthetic).

[0093] In a particular embodiment, the decreased sensitivity is in respect of ETV. Alternatively, the decreased sensitivity is in respect of ADV or LMV. Alternatively, the decreased sensitivity is in respect of TFV. Alternatively, the decreased sensitivity is in respect of FTC. Alternatively, the decreased sensitivity is in respect of ETV and optionally ADV and LMV. Alternatively, the decreased sensitivity is in respect of ADV and TFV and optionally ETV. Alternatively, the decreased sensitivity is in respect of LMV and TFV and optionally ETV. Alternatively, the decreased sensitivity is in respect of ADV and FTC and optionally ETV. Alternatively, the decreased sensitivity is in respect to FTC and TFV and optionally ETV. Alternatively, the decreased sensitivity is in respect of FTC and LMV and optionally ETV. Alternatively, the decreased sensitivity is in respect of ADV and LMV and TFV and optionally ETV. Alternatively, the decreased sensitivity is in respect to ADV and TFV and FTC and optionally ETV. Alternatively, the decreased sensitivity is in respect to LMV and TFV and FTC and optionally ETV. Alternatively, the decrease sensitivity is in respect of ADV and LMV and FTC and optionally ETV. Alternatively, the decreased sensitivity is in respect of ADV and FTC and TFV and LMV and optionally ETV.

[0094] Reference herein to "anti-HBV agents" includes nucleoside and nucleotide analogs as well as immunological reagents (e.g. antibodies to HBV surface components) and chemical, proteinaceous and nucleic acid agents which inhibit or otherwise interfere with viral replication, maintenance, infection, assembly or release. Reference herein to "nucleic acid" includes reference to a sense or antisense molecule, RNA or DNA, oligonucleotides and RNAi and siRNA molecules and complexes containing same.

[0095] In addition to a mutation in the gene encoding DNA polymerase, due to the overlapping nature of the HBV genome (FIG. 1), a corresponding mutation may also occur in the gene encoding the S gene encoding the surface antigen (HBsAg) resulting in reduced interactivity of immunological reagents such as antibodies and immune cells to HBsAg. The reduction in the interactivity of immunological reagents to a viral surface component generally includes the absence of immunological memory to recognize or substantially recognize the viral surface component. The present invention extends, therefore, to an HBV variant exhibiting decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof or a reduced interactivity of an immunological reagent to HBsAg wherein the variant is selected for following ADV and/or LMV combination or sequential treatment. The term "sequential" in this respect means ADV followed by LMV and/or TFV and/or ETV, and/or FTC, LMV followed by ADV and/or TFV and/or FTC and/or ETV or ETV followed by one or more of ADV, FTC, LMV and/or TFV, or multiple sequential administrations of each of ETV, ADV, LMV and/or TFV and/or FTC.

[0096] A viral variant may, therefore, carry a mutation only in the DNA polymerase gene or both in the DNA polymerase gene and the S gene. The term "mutation" is to be read in its broadest context and includes multiple mutations.

[0097] A mutation in any domain of the HBV DNA polymerase and in particular regions F and G, and domains A through to E is contemplated herein provided the mutation leads to decreased sensitivity to ADV and/or LMV and/or TFV and/or ETV and/or FTC and/or LdT or combinations thereof.

[0098] In this specification, reference is particularly made to the conserved regions of the DNA polymerase as defined by domains A to E. Regions A to E are defined by the amino acid sequence set forth in Formula II in Australian Patent No. 734831.

[0099] Particularly, the mutation results in an altered amino acid sequence in any one or more of domains F and G, and domains A through to E or regions proximal thereto of the HBV DNA polymerase.

[0100] Another aspect provides an HBV variant comprising a mutation in an overlapping open reading frame in its genome wherein said mutation is in a region defined by one or more of domains F and G, and domains A through to E of HBV DNA polymerase and wherein said variant exhibits decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0101] A further aspect contemplates an HBV variant comprising a mutation in the nucleotide sequence encoding HBsAg resulting in an amino acid addition, substitution and/or deletion in said HBsAg wherein said variant exhibits decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0102] More particularly, a variant HBV is provided comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to a surface antigen from a reference or wild-type HBV and wherein an antibody generated to the reference or wild-type surface antigen exhibits reduced capacity for neutralizing said HBV variant, said variant selected by exposure of a subject to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0103] The term "combination therapy" means that both combinations of ADV, LMV, FTC, LdT, TFV, and/or ETV are co-administered in the same composition or simultaneously in separate compositions. The term "sequential therapy" means that the two agents are administered within seconds, minutes, hours, days or weeks of each other and in either order. Sequential therapy also encompasses completing a therapeutic course with one or other of ADV, LMV, FTC, TFV, LdT or ETV and then completing a second or third or subsequent therapeutic courses with the other of ADV, LMV, FTC, TFV, LdT or ETV.

[0104] Accordingly, another aspect of contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0105] A further aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to LMV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0106] Yet another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to FTC therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0107] Still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0108] Yet still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0109] Still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant BBV is selected for by exposure of a subject to LdT therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0110] Even yet another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV and LMV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0111] Even still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0112] Yet still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV and ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0113] A further aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to LMV and TFV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0114] Yet a further aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to LMV and ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0115] Another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV and FTC therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0116] Yet another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to TFV and FTC therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0117] Still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to TFV and ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0118] Still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to FTC and LMV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0119] Even another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to FTC and ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0120] Even yet another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV and TFV and/or ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0121] Even still another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV and TFV and/or ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0122] A further aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV and FTC and/or ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0123] Another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to FTC, LMV and TFV and/or ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0124] Yet another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, FTC and TFV and/or ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0125] Still yet another aspect contemplates an HBV variant comprising a surface antigen having an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or truncation compared to the pretreatment HBV and wherein the surface antigen of the variant HBV exhibits an altered immunological profile compared to the pretreatment HBV where the said variant HBV is selected for by exposure of a subject to ADV, LMV, FTC and TFV and/or ETV therapy or therapy by one or more other nucleoside or nucleotide analogs or other anti-HBV agents.

[0126] Particularly, the variants are in isolated form such that they have undergone at least one purification step away from naturally occurring body fluid. Alternatively, the variants may be maintained in isolated body fluid or may be in DNA form. The present invention also contemplates infectious molecular clones comprising the genome or parts thereof from a variant HBV. Furthermore, the present invention provides isolated components from the variant HBVs such as but not limited to an isolated HBsAg. Accordingly, the present invention provides an isolated HBsAg or a recombinant form thereof or derivative or chemical equivalent thereof, said HBsAg being from a variant HBV selected by exposure of a subject to one or more of ADV, LMV, FTC, TFV and/or ETV or optionally one or more nucleoside or nucleotide analogs or other anti-HBV agents.

[0127] More particularly, an isolated variant HBsAg or a recombinant or derivative form thereof or a chemical equivalent thereof is provided wherein said HBsAg or its recombinant or derivative form or its chemical equivalent exhibits an altered immunological profile compared to an HBsAg from a reference HBV, said HBsAg being from a variant HBV selected by exposure of a subject to one or more of ADV, LMV, FTC, TFV and/or ETV or optionally one or more nucleoside or nucleotide analogs or other anti-HBV agents.

[0128] Even more particularly, an isolated variant HBsAg or a recombinant or derivative form thereof or a chemical equivalent thereof is provided wherein said HBsAg or its recombinant or derivative form or its chemical equivalent comprises an amino acid sequence with a single or multiple amino acid substitution, addition and/or deletion or a truncation compared to an HBsAg from a reference HBV and wherein a neutralizing antibody directed to a reference HBV exhibits no or reduced neutralising activity to an HBV carrying said variant HBsAg, said HBsAg being from a variant HBV selected by exposure of a subject to one or more of ADV, LMV, FTC, TFV and/or ETV or optionally one or more nucleoside or nucleotide analogs or other anti-HBV agents.

[0129] Particular mutations in the HBV DNA polymerase include variants selected from patients with HBV recurrence following ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof. Nucleoside or nucleotide analogs or other anti-HBV agents may be indicated during, after or prior to a transplantation procedure (e.g. bone marrow transplantation (BMT) or OLT) or following treatment of patients diagnosed with hepatitis. Following selection of variants, viral loads are obtainable at levels similar to pre-treatment levels or increase while on therapy.

[0130] Useful mutants in the rt region include, in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation.

[0131] Particularly useful mutants are co-mutations at codons 180, 184 and 204 such as rtL180M and rtT184S and rtM204V, or co-mutations at codons 85, 180, 184, 202, 204 and 236 such as rtS85T, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN. Alternative useful mutations contemplated herein include rtP170H, rtT184A, rtY221H/Y, rtI233T/I, rtT240N/T, rtK241R, rtR242K/R, rtW243R/W, rtI253V, rtI254I/F, rtS256R/S rtW257G/W, rtY257H or mutations at codon 239. A "co-mutation" means that a variant will comprise mutations at all mentioned codons. The present invention is particularly directed to co-mutations at codons 180, 184 and 204 as well as co-mutations at codons 85, 180, 184, 202, 204 and 236 but does not extend to a double mutation at codons 180 and 204 alone.

[0132] Such HBV variants are proposed to exhibit a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. It should be noted that the nomenclature system for amino acid positions is based on the methionine residues in the YMDD motif being designated codon rtM204. This numbering system is different to that in Australian Patent No. 734831 where the methionine residue in the YMDD motif within the polymerase gene is designated codon 550. In this regard, rtL180M and rtM204V correspond to L526M and M550V, respectively, in Australian Patent No. 734831. Corresponding mutations may also occur in envelope genes such as in one or more of PreS1, PreS2 and S.

[0133] Another potential mode of action of ADV and other acyclic nucleoside phosphonates is that of immune stimulation (Calio et al, Antiviral Res. 23:77-89, 1994). A number of mutations resulted in changes in the envelope gene detected in HBV variants which may be associated with immune escape. These changes include in one embodiment include sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL162I and sI195M or a combination thereof or an equivalent mutation.

[0134] Particularly useful mutants are sI110V, sP120Q, sF134L/F, sL162I, sL173F, sL175F, sL176V and sP178P/L.

[0135] The identification of the variants herein permits the generation of a range of assays to detect such variants. The detection of such variants may be important in identifying resistant variants to determine the appropriate form of chemotherapy and/or to monitor vaccination protocols, or develop new or modified vaccine preparations.

[0136] Still another aspect contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and G, and A domains through to E or a region proximal thereto of said DNA polymerase and associated with resistance or decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents wherein the presence of such a mutation is an indication of the likelihood of resistance to said ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents.

[0137] Accordingly, another aspect provides a method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside or nucleotide analog selected from ADV, LMV, TFV, LdT, FTC and ETV or optionally other nucleoside or nucleotide analogs, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and A through E or a region proximal thereto of said DNA polymerase and associated with resistance or decreases sensitivity to one or more of ADV, LMV, TFV, LdT, FTC and/or ETV wherein the presence of such a mutation is an indication of the likelihood of resistance to said one or more of ADV, LMV, TFV, LdT, FTC and/or ETV.

[0138] Particularly, the assay detects one or more of the following mutations: in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rt1233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI2541/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181AN and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS2021, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rt1233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation is indicative of a variant wherein said variant exhibits a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof.

[0139] Accordingly, another aspect produces a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other anti-HBV agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase and/or a corresponding region of the S gene, wherein the presence of a mutation selected from, in one embodiment include sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI2081/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL1621 and sI195M or a combination thereof or an equivalent mutation, in even still another embodiment one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rt1233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA811V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI2541/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS2021, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM2041/M, rt1233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or combinations thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0140] A further aspect produces a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other anti-HBV agents, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding the DNA polymerase and/or a corresponding region of the S gene, wherein the presence of a mutation selected from, in one embodiment include s192T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL1621 and sI195M or a combination thereof or an equivalent mutation, in even still another embodiment, in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or combinations thereof or an equivalent one or more other mutation is indicative of a variant which exhibits a decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0141] The detection of HBV or its components in cells, cell lysates, cultured supernatant fluid and bodily fluid may be by any convenient means including any nucleic acid-based detection means, for example, by nucleic acid hybridization techniques or via one or more polymerase chain reactions (PCRs). The term "bodily fluid" includes any fluid derived from the blood, lymph, tissue or organ systems including serum, whole blood, biopsy and biopsy fluid, organ explants and organ suspension such as liver suspensions. The invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others. Other forms of detection include Northern blots, Southern blots, PCR sequencing, antibody procedures such as ELISA, Western blot and immunohistochemistry. A particularly useful assay includes the reagents and components required for immobilized oligonucleotide- or oligopeptide-mediated detection systems.

[0142] One particularly useful nucleic acid detection system is the reverse hybridization technique. In this technique, DNA from an HBV sample is amplified using a biotin or other ligand-labeled primer to generate a labeled amplificon. Oligonucleotides immobilized to a solid support such as a nitrocellulose film are then used to capture amplified DNA by hybridization. Specific nucleic acid fragments are identified via biotin or the ligand. Generally, the labeled primer is specific for a particular nucleotide variation to be detected. Amplification occurs only if the variation to be detected is present. There are many forms of the reverse hybridization assay and all are encompassed by the present invention.

[0143] Another aspect contemplated herein provides a method for detecting a variant HBV exhibiting an altered immunological profile said method comprising isolating an HBV from a subject exposed to a nucleoside or nucleotide analog selected from the listed consisting of ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof, and then contacting said HBV with a panel of one or more antibodies to a surface antigen and screening for any change in binding affinity or binding spectrum.

[0144] In a related embodiment, a method is contemplated for detecting a variant HBV exhibiting an altered immunological profile said method comprising isolating a serum sample from a subject exposed to a nucleoside or nucleotide analog selected from the listed consisting of ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof, and then contacting the serum with a panel of HBV surface antigens or antibody-binding fragments thereof and screening for any change in binding affinity or binding spectrum.

[0145] Detecting HBV replication in cell culture is particularly useful.

[0146] This and other aspects are particularly amenable to microarray analysis such as to identify oligonucleotides including sense and antisense molecules, RNAi or siRNA molecules or DNA or RNA-binding molecules which down-regulate genomic sequences or transcripts of HBV. Microarray analysis may also be used to identify particular mutations in the HBV genome such as within the HBV DNA polymerase-coding region or the HBsAg-coding region.

[0147] Another aspect of contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV by: generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in a plasmid vector and then transfecting said cells with said construct; contacting the cells, before, during and/or after transfection, with the agent to be tested; culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agents; and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

[0148] In a particular embodiment, the plasmid vector may include genes encoding part or all of other viral vectors such as baculovirus or adenovirus (Ren and Nassal, 2001, supra) and the method comprises: generating a genetic construct comprising a replication competent-effective amount of the genome from the HBV contained in or fused to an amount of a baculovirus genome or adenovirus genome effective to infect cells and then infecting said cells with said construct; contacting the cells, before, during and/or after infection, with the agent to be tested; culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and then subjecting the Cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

[0149] In an alternative embodiment, the method comprises: generating a continuous cell line comprising an infectious copy of the genome of the HBV in a replication competent effective amount such that said infectious HBV genome is stably integrated into said continuous cell line such as but not limited to 2.2.15 or AD; contacting the cells with the agent to be tested; culturing the cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to the agent; and then subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of the agent.

[0150] The above-mentioned methods are particularly useful in identifying or developing agents against HBV variants such as those carrying mutations, in one embodiment includes rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation; in a further embodiment includes sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL162I and sI195M or a combination thereof or an equivalent mutation.

[0151] Accordingly, another aspect contemplates a method for determining whether an HBV strain exhibits reduced sensitivity to a nucleoside or nucleotide analog or other potential anti-HBV agent, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence of the envelope genes or DNA polymerase gene selected from, in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation; in a further embodiment, in one embodiment include sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY2251I/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL1621 and sI195M or a combination thereof or an equivalent mutation and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof.

[0152] The detection of amino acid variants of DNA polymerase is conveniently accomplished by a range of amino acid detection techniques. Where an HBV variant comprises an amino acid change, then such an isolate is considered a putative HBV variant having an altered DNA polymerase activity.

[0153] Further contemplated herein are agents which inhibit ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof resistant HBV variants. Such agents are particularly useful if long term treatment by ADV, LMV, FTC, TFV, LdT and/or ETV and/or optionally other nucleoside or nucleotide analogs such as TFV is contemplated by the clinician. The agents may be DNA or RNA or proteinaceous or non-proteinaceous chemical molecules. Natural product screening such as from plants, coral and microorganisms is also contemplated as a useful potential source of masking agents as is the screening of combinatorial or chemical libraries. The agents may be in isolated form or in the form of a pharmaceutical composition or formulation and may be administered in place of or sequentially or simultaneously with a nucleoside or nucleotide analog. Furthermore, rationale drug design is contemplated including solving the crystal or NMR structure of, for example, HBV DNA polymerase and designing agents which can bind to the enzyme's active site. This approach may also be adapted to other HBV components.

[0154] Accordingly, another aspect contemplates a method for detecting an agent which exhibits inhibitory activity to an HBV which exhibits resistance or decreased sensitivity ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof, said method comprising: generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in a plasmid vector and then transfecting said cells with said construct; contacting said cells, before, during and/or after transfection, with the agent to be tested; culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.

[0155] Still another aspect provides a method for detecting an agent which exhibits inhibitory activity to an HBV which exhibits resistance or decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof, method comprising: generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct; contacting said cells, before, during and/or after infection, with the agent to be tested; culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.

[0156] Particularly, the HBV genome is stably integrated into the cells' genome.

[0157] Useful cells include 2.2.15 cells (Price et al, Proc. Natl. Acad. Sci. USA 86(21):8541-8544, 1989 or AD cells (also known as HepAD32 cells or HepAD79 cells [Ying et al, 2000, supra].

[0158] Whilst the baculovirus vector is a particularly useful in the practice of the instant method a range of other vectors may also be used such as but not limited to adenoviral vectors.

[0159] Cell lines (e.g. 2.2.15 or AD cells) carrying genetic constructs comprising all or a portion of an HBV genome or a gene or part of a gene therefrom are also contemplated herein.

[0160] Also provided is the use of the subject HBV variants to screen for anti-viral agents. These anti-viral agents inhibit the virus. The term "inhibit" includes antagonizing or otherwise preventing infection, replication, assembly and/or release or any intermediate step. Particular anti-viral agents include nucleoside or nucleotide analogs or anti-HBV agents, as well as non-nucleoside molecules.

[0161] In addition, rational drug design is also contemplated to identify or generate chemical molecules which either mimic a nucleoside or which interact with a particular nucleotide sequence or a particular nucleotide. Combinatorial chemistry and two hybrid screening are some of a number of techniques which can be employed to identify potential therapeutic or diagnostic agents.

[0162] In one example, the crystal structure or the NMR structure of polymerase or the surface antigen is used to rationally design small chemical molecules likely to interact with key regions of the molecule required for function and/or antigenicity. Such agents may be useful as inhibitors of polymerase activity and/or may alter an epitope on the surface antigen.

[0163] Several models of the HBV polymerase have been prepared due to the similarity with reverse transcriptase from HIV (Das et al, J. Virol. 75(10:4771-4779, 2001; Bartholomeusz et al, Intervirology 40(5-6):337-342 1997; Allen et al, Hepatology 27(6):1670-1677, 1998). The models of the HBV polymerase can be used for the rational drug design of new agents effective against HBV encoding the resistant mutations as well as wild type virus. The rational drug that is designed may be based on a modification of an existing antiviral agent such as the agent used in the selection of the HBV encoding the mutations associated with resistance. Viruses or clones expressing HBV genomic material encoding the mutations may also be used to screen for new antiviral agents.

[0164] In an alternative embodiment, a method is contemplated for detecting an agent which exhibits inhibitory activity to an HBV polymerase in an in vitro polymerase assay. The HBV polymerase activity can be examined using established assays (Gaillard et al, 2002, supra; Xiong et al, 1998, supra).

[0165] As indicated above, microarray technology is also a useful means of identifying agents which are capable of interacting with defined HBV internal or external components. For example, arrays of HBV DNA polymerase or peptide fragments thereof carrying different amino acid variants may be used to screen for agents which are capable of binding or otherwise interacting with these molecules. This is a convenient way of determining the differential binding patterns of agents between HBV variants. Arrays of antibodies may also be used to screen for altered HBsAg molecules. Microarrays are also useful in proteomic analysis to identify molecules such as antibodies, interferons or cytokines which have an ability to interact with an HBV component. Microarrays of DNA and RNA molecules may also be employed to identify sense and antisense molecules for genetic regions on the HBV genome or transcripts thereof.

[0166] The above methods are particularly useful in identifying an inhibitor of an HBV resistant to or exhibiting reduced sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof. The present invention extends, therefore, to compositions of the inhibitors. The inhibitors may also be in the form of antibodies or genetic molecules such as ribozymes, antisense molecules and/or sense molecules for co-suppression or the induction of RNAi or may be other nucleoside or nucleotide analogs or other anti-HBV agents or derivatives of known analogs. Reference to RNAi includes reference to short, interfering RNAs (siRNA) and all RNAi-type molecules may be DNA-derived or synthetic.

[0167] The term "composition" includes a "pharmaceutical composition" or a formulation.

[0168] The inhibitor is referred to below as an "active ingredient" or "active compound" and may be selected from the list of inhibitors given above.

[0169] The composition may include an antigenic component of the HBV, a defective HBV variant or an agent identified through natural product screening or rational drug design (including combinatorial chemistry).

[0170] Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0171] The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of encoding an aspartyl protease inhibitor. The vector may, for example, be a viral vector.

[0172] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0173] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the active ingredient and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and the freeze-drying technique which yield a powder of active ingredient plus any additionally desired ingredient.

[0174] When the active ingredient is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1μg and 200 mg of active compound. Alternative dosage amounts include from about 1μg to about 1000 mg and from about 10μg to about 500 mg. These dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year.

[0175] The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavouring. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

[0176] As stated above, the present invention further extends to an isolated HBsAg from the HBV variants herein described. More particularly, the present invention provides an HBsAg or a recombinant form thereof or derivative or chemical equivalent thereof. The isolated surface component and, more particularly, isolated surface antigen or its recombinant, derivative or chemical equivalents are useful in the development of biological compositions such as vaccine formulations.

[0177] Yet another aspect provides a composition comprising a variant HBV resistant to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or an HBV surface antigen from said variant HBV or a recombinant or derivative form thereof or its chemical equivalent and one or more pharmaceutically acceptable carriers and/or diluents. Such a composition may be regarded as a therapeutic composition and is useful in generating an immune response including a humoral response. Generally, the HBV variants are "defective" and in themselves are unable to cause a sustained infection in a subject.

[0178] As indicated above, antibodies may be generated to the mutant HBV agents and used for passive or direct vaccination against infection by these viruses. The antibodies may be generated in humans or non-human animals. In the case of the latter, the non-human antibodies may need to be deimmunized or more specifically humanized prior to use. Deimmunized may include, for example, grafting complimentarity determining regions (CDRs) from the variable region of a murine or non-human animal anti-HBV antibody onto a human consensus fragment antibody binding (Fab) polypeptide. Alternatively, amino acids defining epitopes in the variable region of the antibody may be mutated so that the epitopes are no longer recognized by the human MHC II complex.

[0179] Insofar as ribozyme, antisense or co-suppression (RNAi) or siRNA or complexes thereof repression is concerned, this is conveniently aimed at post-transcription gene silencing. DNA or RNA may be administered or a complex comprising RNAi or a chemical analog thereof specific for HBV mRNA may be employed.

[0180] All such molecules may be incorporated into pharmaceutical compositions.

[0181] In another embodiment, provided is a biological composition comprising a variant HBV or an HBsAg or L, M or S proteins from said variant HBV or a recombinant or derivative form thereof or its chemical equivalent.

[0182] Generally, if an HBV is used, it is first attenuated. The biological composition generally further comprises one or more pharmaceutically acceptable carriers and/or diluents.

[0183] The biological composition may comprise HBsAg or like molecule from one HBV variant or the composition may be a cocktail of HbsAgs or L, M or S proteins or like molecules from a range of ADV- and/or LMV- and/or, FTC-LdT-, and/or TFV-resistant HBV variants. Similar inclusions apply where the composition comprises an HBV.

[0184] Further provided is the use of defective HBV variants in the manufacture of therapeutic vaccines to vaccinate individuals against infection by HBV strains having a particular nucleotide sequence or encoding a particular polymerase or surface antigen or L, M or S proteins.

[0185] Examples of suitable vaccine candidates are defective forms of HBV variants comprising a mutation selected from, in one embodiment includes, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation; in a further embodiment, sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL162I and sI195M or a combination thereof or an equivalent mutation.

[0186] In one embodiment, for example, an HBV variant may be identified having a particular mutation in its polymerase conferring resistance or decreased sensitivity to a nucleoside or nucleotide analog. This variant may then be mutated to render it defective, i.e. attenuated or unable to cause infection. Such a defective, nucleoside or nucleotide analog-resistant virus may then be used as a therapeutic vaccine against virulent viruses having the same mutation in its polymerase.

[0187] The subject invention extends to kits for assays for variant HBV resistant to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof. Such kits may, for example, contain the reagents from PCR or other nucleic acid hybridization technology or reagents for immunologically based detection techniques. A particularly useful assay includes the reagents and components required for immobilized oligonucleotide- or oligopeptide-mediated detection systems.

[0188] Still another aspect of contemplates a method for determining the potential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combination thereof, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for a mutation in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in any one or more of domains F and G, and domains A through to E or a region proximal thereto of said DNA polymerase and associated with resistance or decreased sensitivity to ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof, wherein the presence of such a mutation is an indication of the likelihood of resistance to said ADV, LMV, TFV, LdT, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionally other nucleoside or nucleotide analogs or other anti-HBV agents or combinations thereof.

[0189] An assessment of a potential viral variant is important for selection of an appropriate therapeutic protocol. Such an assessment is suitably facilitated with the assistance of a computer programmed with software, which inter alia adds input codes for at least two features associated with the viral variants to provide a value corresponding to the resistance or sensitivity of a viral variant to a particular chemical compound or immunological agent. The value can be selected from (a) the ability to exhibit resistance for reduced sensitivity to a particular compound or immunological agent; (b) an altered DNA polymerase from wild-type HBV; (c) an altered surface antigen from wild-type HBV; or (d) morbidity or recovery potential of a patient. Thus, in accordance with the present invention, the values for such features are stored in a machine-readable storage medium, which is capable of processing the data to provide a value for a particular viral variant or a biological specimen comprising same.

[0190] Thus, in another aspect, a computer program product is contemplated for assessing the likely usefulness of a viral variant or biological sample comprising same for determining an appropriate therapeutic protocol in a subject (FIG. 15), said product comprising: (1) code that receives as input code for at least two features associated with said viral agents or biological sample comprising same, wherein said features are selected from: (a) the ability to exhibit resistance for reduced sensitivity to a particular compound or immunological agent; (b) an altered DNA polymerase from wild-type HBV; (c) an altered surface antigen from wild-type HBV; or (d) morbidity or recovery potential of a patient; (2) code that adds said input code to provide a sum corresponding to a value for said viral variants or biological samples; and (3) a computer readable medium that stores the codes.

[0191] In a related aspect, a computer is provided for assessing the likely usefulness of a viral variant or biological sample comprising same in a subject, wherein said computer comprises: (1) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said machine-readable data comprise input codes for at least two features associated with said viral variant or biological sample; wherein said features are selected from:--(a) the ability to exhibit resistance for reduced sensitivity to a particular compound or immunological agent; (b) an altered DNA polymerase from wild-type HBV; (c) an altered surface antigen from wild-type HBV; or (d) morbidity or recovery potential of a patient; (2) a working memory for storing instructions for processing said machine-readable data; (3) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine readable data to provide a sum of said input code corresponding to a value for said compound(s); and (4) an output hardware coupled to said central processing unit, for receiving said value.

[0192] Any general or special purpose computer system is contemplated by the present invention and includes a processor in electrical communication with both a memory and at least one input/output device, such as a terminal. FIG. 15 shows a generally suitable computer system. Such a system may include, but is not limited, to personal computers, workstations or mainframes. The processor may be a general purpose processor or microprocessor or a specialized processor executing programs located in RAM memory. The programs may be placed in RAM from a storage device, such as a disk or pre-programmed ROM memory. The RAM memory in one embodiment is used both for data storage and program execution. The computer system also embraces systems where the processor and memory reside in different physical entities but which are in electrical communication by means of a network.

[0193] In an alternative embodiment, the program screens for a mutation selected from, in one embodiment, rtS256R/S and rtW257G/W, in another embodiment includes rtH9D, rtL180M, rtM204V, rtT184S and rtN238T, or yet another embodiment includes rtQ215S, rtR242K/R, and rtW243R/W, or yet another embodiment includes rtN118S, rtR120R, rtT184A and rtY257H, or still another embodiment includes rtH216H/P, rtL229M/L, rtI233T/I, rtT240N/T, rtK241R and rtH248N or yet another embodiment includes rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F, or still another embodiment includes rtY221H/Y, rtV214A, rtA181A/V and rtN236T, or still another embodiment includes rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/DN, or yet another embodiment includes rtF61L, rtS78T, rtV84MN, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S, or finally in another embodiment includes rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V, or a combination thereof or an equivalent mutation; in a further embodiment sI92T/I, sL175F and sI195M, in another embodiment include sS207R, or yet another embodiment include sI110V and sP120Q, or yet another embodiment include sF80S, sI208I/L, sS210K, sF220L/F and sY225H/Y, or yet another embodiment includes sF134L/F and sL173F, or yet another embodiment includes sC76Y and sL173F, or yet another embodiment includes sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M, or yet another embodiment includes sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sWl96stop/W, s210R/S and sY225H/Y, and finally in another embodiment includes sL20F, sI42L, sV144D, sL162I and sI195M or a combination thereof or an equivalent mutation.

[0194] The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

[0195] Overlapping Genome of HBV

[0196] The overlapping genome of HBV is represented in FIG. 1. The gene encoding DNA polymerase (P), overlaps the viral envelope genes, Pre-S 1 and Pre-S2, and partially overlaps the X and core (C) genes. The HBV envelope comprises small, middle and large proteins HBV surface antigens. The large protein component is referred to as the HBV surface antigen (HBsAg) and is encoded by the S gene sequence. The Pre-S 1 and Pre-S2 gene sequences encode the other envelope components.

EXAMPLE 2

[0197] Patients on TFV and LMV and Analysis of HBV DNA

[0198] Patient A: During TFV and LMV treatment, unique HBV mutations were detected by sequencing (Table 4). This patent has had selected unique mutations at rtS256R/S and rtW257G/W (Table 4, FIGS. 4, 5, and 6) during virological breakthrough on treatment. These changes do not alter the HBsAg in the overlapping reading frame as they are after the termination codon in the HBsAg reading frame.

[0199] Patient B: During TFV and LMV treatment, unique HBV mutations were detected by sequencing (Table 5 and FIGS. 7A, 7B, 8, and 9). The unique changes in the rt region of the HBV DNA polymerase include rtL180M+rtM204V previously demonstrated to be associated with LMV resistance in_conjunction with a mutation at codon 184 (rtT184S) and rtH9D and rtN238T. The change at codon 184 in the rt has been previously noted with ETV+/-LMV resistance (Tenney et al, Antimicrob Agents Chemother 48(9):3498-507, 2004) but has not been reported with TFV+LMV resistance and virological breakthrough during treatment.

[0200] The changes in the HBsAg while on TFV and LMV treatment include sI92T/I, sL175F and sI195M. The last two changes correspond to changes in the polymerase at rtT184S and rtM204V, respectively.

[0201] Patient C: The HBV mutations during LMV and TFV treatment are listed in Table 6 and FIGS. 10, 11, 12. The unique changes in the rt region of the HBV DNA polymerase include rtR242K/R, W243R/W and rtQ215S. The changes in the HBsAg while on LMV and TFV treatment include sS207R.

[0202] Patient D: The HBV mutations during LMV and TFV treatment are listed in Table 7 and FIGS. 13, 14, 15. The unique changes in the rt region of the HBV DNA polymerase include rtT184A and rtY257H other changes include rtN118S and rtR102E.

[0203] The changes in the HBsAg while on LMV and TFV treatment include sI110V and sP120Q.

EXAMPLE 3

[0204] Patients on Antiviral Therapy and Analysis of HBV DNA

[0205] Treatment of patients with chronic hepatitis B virus with nucleos(t)ide analogs can result in the selection of HBV variants encoding mutations that may be associated with reduced sensitivity to the antiviral agent.

[0206] Resistance to ADV has been associated with a mutations in the putative tri-phosphate binding site at rtN236T (Angus et al, Gastroenterolog. 125(2):292-297, 2003). Therefore other mutations in this region selected by ADV or other antiviral treatments may be important in antiviral resistance against ADV and/or other nucleos(t)ide treatments. Important codons include 239 which is highly conserved, 240, 241, 242 and 243.

[0207] In addition to the mutation at rtN236T, other mutations may increase resistance and/or replication. Once such mutation is at codon 221

[0208] Patient E: The HBV mutations during LMV treatment are listed in Table 8 and FIGS. 16, 17, and 18. The unique changes in the rt region of the HBV DNA polymerase include rtT240N/T and rtK241R. Other important unique changes include rtH216H/P, rtL229M/L, rtI233T.

[0209] The changes in the HBsAg while on LMV treatment include sF80S, sI208UL, sS210K, sF220L/F and sY225H/Y.

[0210] Patient F: The HBV mutations during LMV treatment are listed in Table 9 and FIGS. 19, 20, and 21. The unique changes in the rt region of the HBV DNA polymerase include, rtR242R/K and rtW243G/W. Other important unique changes include rtL180M, rtA181T, rtH216H/P and rtE218D/E, refer to Table 9 for all other changes.

[0211] The important changes in the HBsAg in or near the "a" determinant while on LMV treatment include sF134L/F and sL173F (refer to Table 9 for all other changes).

[0212] Patient G: The HBV mutations during LMV treatment are listed in Table 10 and FIGS. 22, 23 and 24. The unique changes in the rt region of the HBV DNA polymerase include rtY221H/Y. Other important unique changes include rtV214A, rtA181A/V and rtN236T. (Refer to Table 9 for all other changes).

[0213] The important changes in the HBsAg in or near the "a" determinant while on LMV treatment include sL173F (refer to Table 9 for all other changes).

[0214] Patient H.

[0215] Patient H has been previously treated with a number of nucleoside/nucleotide analogs and was resistant individually to LMV, then ETV, then ADV. The patient was subsequently treated with combination LMV and ADV and ETV and is now resistant to all these agents. The HBV mutations during treatment are listed in FIGS. 25, 26 and 27.

[0216] This patient has selected unique combinations of mutations rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V, and rtN236I/DN.

[0217] This includes the known LMV resistant mutations at rtL180M and rtM204V also the known ETV resistant mutations at rtL180M, rtT184G, rtS202I, and rtM204V. This patient has selected unique mutations at codon 236 at rtN236I/DN that have not previously been reported with ADV resistance. Together all these mutations may be important for the combined ADV, LMV and ETV resistance mutations.

[0218] Changes in the HBsAg include sD33D/E, sC69 Stop, sC76 Stop, sR79H, sP120T, sL176V, sV194F and sI195M.

[0219] Patient I

[0220] Patient I was previously treated with LMV, then ADV. This patient is now being treated with TFV and LMV and has selected mutations which may be associated with reduced sensitivity to these agents. The HBV mutations during TDF and LMV treatment are listed in FIGS. 28, 29 and 30.

[0221] This includes the mutations at rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/PN/S. In particular the mutation at rtI233T/I was not detected previously in HBV isolated from this patient pre-TFV treatment.

[0222] Changes in the HBsAg include sC69Stop, sM75I/M, sY134N, sK141K/E, sP178P/L, sW196stop/W, s210R/S, and sY225H/Y.

[0223] Patient J

[0224] Patient J was previously treated with LMV and selected HBV with mutations associated with LMV resistance this includes the polymerase mutations at rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V and the envelope mutations at sL20F, sI42L, sV144D, sL162I and sI195M.

[0225] This patient was subsequently treated with ADV and did not respond and then ETV and also did not respond. This suggests that this patient may have selected mutations during LMV treatment that may have affected the subsequent antiviral non-response or primary resistance to ADV and then ETV.

[0226] In particular the mutations at rtI253V and rtP170H may be important for the primary resistance to ADV and ETV.

EXAMPLE 4

[0227] Detection of Viral Markers

[0228] Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), anti-HBe and hepatitis B core antigen (HBcAg) specific IgG and IgM were measured using commercially available immunoassays (Abbott Laboratories, North Chicago, Ill., USA). Hepatitis B viral DNA levels were measured using a capture hybridization assay according to the manufacturer's directions (Digene Hybrid Capture II, Digene Diagnostics Inc., Beltsville, Md.). The manufacturers stated cut-off for detecting HBV viremia in clinical specimens was 0.7×10⁶ copies/ml or 2.5 pg/ml, [Hendricks et al, Am J Clin Pathol 104:537-46, 1995]. HBV DNA levels can also be quantitated using other commercial kits such as Cobas amplification HBV monitor kit (Roche).

EXAMPLE 5

[0229] Sequencing of HBV DNA

[0230] HBV DNA was extracted from 100 μ1 of serum as described previously by Aye et al, J. Hepatol. 26:1148-1153, 1997. Oligonucleotides were synthesized by Geneworks, Adelaide, Australia. Amplification of the HBV polymerase gene has been described by Aye et al, 1997, supra.

[0231] The specific amplified products were purified using PCR purification columns from MO BIO Laboratories Inc (La Jolla, Calif.) and directly sequenced using Big Dye terminator Cycle sequencing Ready Reaction Kit (Perkin Elmer, Cetus Norwalk, Conn.). The PCR primers were used as sequencing primers, OS1 5'-GCC TCA TTT TGT GGG TCA CCA TA-3' (nt 1408-1430) [SEQ ID NO:1], TTA3 5'-AAA TTC GCA GTC CCC AAA-3' (nt2128-2145) [SEQ ID NO:2], JM 5'-TTG GGG TGG AGC CCT CAG GCT-3' (nt1676-1696) [SEQ ID NO:3], TTA4 5'-GAA AAT TGG TAA CAG CGG-3' (nt 2615-2632) [SEQ ID NO:4], 0S2 5' TCT CTG ACA TAC TTT CCA AT 3' (nt 2798-2817) [SEQ ID NO:5], to sequence the internal regions of the PCR products.

EXAMPLE 6

[0232] Adefovir Dipivoxil

[0233] ADV

[0234] ADV (formerly Bis-pom PMEA)) is a potent inhibitor of HBV replication. The structure of ADV is shown in FIG. 2 and its synthesis is described by Benzaria et al, J Med Chem. 39:4958-4965, 1996.

EXAMPLE 7

[0235] Tenofovir

[0236] TFV

[0237] TFV (formerly Bis-pom PMPA) is a potent inhibitor of HBV replication. The structure of tenofovir is shown in FIG. 3 and its synthesis is described by Srinivas and Fridland, Antimicrob Agents Chemother. 42(6):1484-1487, 1998.

[0238] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

TABLE-US-00004 TABLE 4 Patient A HBV Polymerase and envelope mutations detected duringTFV/LMV therapy Viral Load ALT (IU/ml) (IU/ml) G'type Polymerase # 114 A wildtype 25 PCR-ve 24 PCR-ve 1.79E+07 A S256R/S W257G/w

TABLE-US-00005 TABLE 5 Patient B HBV Polymerase and envelope mutations detected duringTFV/LMV therapy Viral Load ALT Polymerase (IU/ml) (IU/ml) G'type # HBsAG# C H9D I92T/I L180M L175F T184S I195M M204V 1.99E+07 C H9D I92T/I L180M L175F T184S I195M M204V N238T 1.06E+06 C H9D I92T/I L180M L175F T184S I195M M204V N238N/T 1.42E+05 C H9D I92T/I L180M L175F T184S I195I/M M204/V 1.00E+08 C H9D I92T/I L180M L175F T184S I195M M204V N238T

TABLE-US-00006 TABLE 6 Patient C HBV Polymerase and envelope mutations detected duringTFV/LMV therapy Viral Load ALT Polymerase (IU/ml) (IU/ml) G'type # HBsAG# 1.70E+09 71 D V191I/V W182*/W Q215P/S S207R 55 D V191I/V W182*/W Q215P/S S207R 2.82E+05 D Q215S S207R R242K/R W243R/W

TABLE-US-00007 TABLE 7 Patient D HBV Polymerase and envelope mutations detected during TFV and LMV therapy Viral Load ALT Polymerase (IU/ml) (IU/ml) G'type # HBsAG# D N118S I110V R120R P120Q T184A Y257H

TABLE-US-00008 TABLE 8 Patient E HBV Polymerase and envelope mutations detected during LMV therapy Viral Load ALT Polymerase (IU/ml) (IU/ml) G'type # HBsAG# D F122V/F F80S S135Y I208I/L H216H/P S210K L229M/L F220L/F I233T/I Y225H/Y T240N/T K241R

TABLE-US-00009 TABLE 9 Patient F HBV Polymerase and envelope mutations detected during LMV therapy Viral Load ALT Polymerase (IU/ml) (IU/ml) G'type # HBsAG# B N124D F134L/F S135A L173F S143T/S I208I/L L180M S210T/S A181V H216H/P E218D/E H238D R242K/R W243G/W I254I/f

TABLE-US-00010 TABLE 10 Patient G HBV Polymerase and envelope mutations detected during ADV therapy Viral Load ALT Polymerase (IU/ml) (IU/ml) G'type # HBsAG# 1.98E+06 467 B L115M W196S M204I V214A M250I 1.42E+05 PCR-ve 1.61E+04 PCR-ve 2.00E+03 PCR-ve B N53D C76Y L115M L173L/F A181A/V V214A Y221H/Y N236T

BIBLIOGRAPHY

[0239] Allen et al, Hepatology 27(6):1670-1677, 1998

[0240] Angus et al, Gastroenterology. 125(2):292-297, 2003

[0241] Aye et al, J. Hepatol. 26:1148-1153, 1997

[0242] Bartholomeusz et al, Intervirology 40(5-6):337-342 1997

[0243] Benhamou et al, Lancet 358: 718-723, 2001

[0244] Benzaria et al, J Med Chem. 39: 4958-4965, 1996

[0245] Boyd et al, Antiviral Chem Chemother. 32: 358-363, 1987

[0246] Calio et al, Antiviral Res. 23:77-89, 1994

[0247] Das et al, J. Virol. 7500:4771-4779, 2001

[0248] Dienstag et al, New England J Med 333:1657-1661, 1995

[0249] Frick et al, Antimicrob. Agents Chemother. 37:2285-2292, 1993

[0250] Gaillard et al, Antimicrob Agents Chemother. 46(4):1005-1013, 2002

[0251] Gilson et al, J Viral Hepat 6:387-395, 1999

[0252] Heathcote et al, Hepatology 28:A620, 1998

[0253] Hendricks et al, Am J Clin Pathol 104:537-46, 1995

[0254] Kruger et al, Hepatology 22:219A, 1994

[0255] Main et al, J. Viral Hepatitis 3:211-215, 1996

[0256] Norder et al, J. Gen. Virol. 74:341-1348, 1993

[0257] Perrillo et al, Hepatology 32:129-134, 2000

[0258] Peters et al, Transplantation 68:1912-1914, 1999

[0259] Price et al, Proc. Natl. Acad. Sci. USA 86(21):8541-8544, 1989

[0260] Ren and Nassal, J. Virol. 75(3):1104-1116, 2001

[0261] Severini et al, Antimicrobial Agents Chemother. 39:430-435, 1995

[0262] Srinivas and Fridland, Antimicrob Agents Chemother. 42(6):1484-1487, 1998

[0263] Stuyver et al, Hepatology 33:751-757, 2001

[0264] Summers and Mason, Cell 29:403-415, 1982

[0265] Suo et al, J Biol Chem. 273(42):27250-27258. 1998

[0266] Tenney et al, Antimicrob Agents Chemother. 48:3498-507, 2004

[0267] Vere Hodge, Antiviral Chem Chemother 4:67-84, 1993

[0268] Xiong et al, Hepatology. 28(6):1669-1673, 1998

[0269] Ying et al, J Viral Hepat. 7(1):79-83, 2000

[0270] Ying et al, J Viral Hepat. 7(2):161-165, 2000

REFERENCE TO SEQUENCE LISTING

[0271] A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. §1.52(e). The name of the ASCII text file for the Sequence Listing is 13143266₁₃ 1TXT, the date of creation of the ASCII text file is Apr. 19, 2012, and the size of the ASCII text file is 65.3 KB.

Sequence CWU 1

1

32123DNAArtificial SequencePCR primer 1gcctcatttt gtgggtcacc ata 23218DNAArtificial SequencePCR primer 2aaattcgcag tccccaaa 18321DNAArtificial SequencePCR primer 3ttggggtgga gccctcaggc t 21418DNAArtificial SequencePCR primer 4gaaaattggt aacagcgg 18520DNAArtificial SequencePCR primer 5tctctgacat actttccaat 206987DNAHepatitis B Virus 6tccacctcta agagacagtc atcctcaggc catgcagtgg aattccactg ccttccacca 60agctctgcag gatcccagag tcaggggtct gtattttcct gctggtggct ccagttcagg 120aacagtaaac cctgctccga atattgcctc tcacatctcg tcaatctccg cgaggactgg 180ggaccctgtg acgaacatgg agaacatcac atcaggattc ctaggacccc tgctcgtgtt 240acaggcgggg tttttcttgt tgacaagaat cctcacaata ccgcagagtc tagactcgtg 300gtggacttct ctcaattttc tagggggatc acccgtgtgt cttggccaaa attcgcagtc 360cccaacctcc aatcactcac caacctcctg tcctccaatt tgtcctggtt atcgctggat 420gtgtctgcgg cgttttatca tattcctctt catcctgctg ctatgcctca tcttcttatt 480ggttcttctg gattatcaag gtatgttgcc cgtttgtcct ctaattccag gatcaacaac 540aaccagtacg gggccatgca aaacctgcac gactcctgct caaggcaact ctatgtttcc 600ctcatgttgc tgtacaaaac ctacggatgg aaattgcacc tgtattccca tcccatcgtc 660ctgggctttc gcaaaatacc tatgggagtg ggcctcagtc cgtttctctt ggctcagttt 720actagtgcca tttgttcagt ggttcgtagg gctttccccc actgtttggc tttcagctat 780atggatgatg tggtattggg ggccaagtct gtacagcatc gtgagtccct ttataccgct 840gttaccaatt ttcttttgtc tctgggtata catttaaacc ctaacaaaac aaaaagatgg 900ggttattccc taaacttcat gggttacata attggaagkk ggggaacatt gccacaggat 960catattgtac aaaagatcaa acactgt 9877329PRTHepatitis B Virusmisc_feature(313)..(313)Xaa can be Arg or Ser 7Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Phe His 1 5 10 15 Cys Leu Pro Pro Ser Ser Ala Gly Ser Gln Ser Gln Gly Ser Val Phe 20 25 30 Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr 35 40 45 Cys Leu Ser His Leu Val Asn Leu Arg Glu Asp Trp Gly Pro Cys Asp 50 55 60 Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val 65 70 75 80 Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu 85 90 95 Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg 100 105 110 Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn 115 120 125 Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala 130 135 140 Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Leu Ile 145 150 155 160 Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser 165 170 175 Arg Ile Asn Asn Asn Gln Tyr Gly Ala Met Gln Asn Leu His Asp Ser 180 185 190 Cys Ser Arg Gln Leu Tyr Val Ser Leu Met Leu Leu Tyr Lys Thr Tyr 195 200 205 Gly Trp Lys Leu His Leu Tyr Ser His Pro Ile Val Leu Gly Phe Arg 210 215 220 Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gln Phe 225 230 235 240 Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cys Leu 245 250 255 Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Val Gln 260 265 270 His Arg Glu Ser Leu Tyr Thr Ala Val Thr Asn Phe Leu Leu Ser Leu 275 280 285 Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Ser Leu 290 295 300 Asn Phe Met Gly Tyr Ile Ile Gly Xaa Xaa Gly Thr Leu Pro Gln Asp 305 310 315 320 His Ile Val Gln Lys Ile Lys His Cys 325 8291PRTHepatitis B Virus 8Pro Pro Leu Arg Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr 1 5 10 15 Ala Phe His Gln Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe 20 25 30 Pro Ala Gly Gly Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile 35 40 45 Ala Ser His Ile Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr 50 55 60 Asn Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu 65 70 75 80 Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser 85 90 95 Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Val 100 105 110 Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr 115 120 125 Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg 130 135 140 Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu 145 150 155 160 Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro 165 170 175 Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro 180 185 190 Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr 195 200 205 Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala 210 215 220 Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu 225 230 235 240 Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp 245 250 255 Leu Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser 260 265 270 Ile Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp 275 280 285 Val Tyr Ile 290 93197DNAHepatitis B Virus 9atgcccctat cttatcaaca cttccggaaa ctactgttgt tagacgacga ggcaggtccc 60ctagaagaag aactccctcg cctcgcagac gaaggtctca atcgccgcgt cgcagaagat 120ctaaatctcg ggaatctcaa tgttagtatc ccttggactc ataaggtggg aaactttact 180gggctttatt cttctactgt acctgtcttt aatcctgact ggcaaactcc ctcttttcct 240cacattcatt tgaaagagga tattattgat agatgtcaac aatatgtagg ccctcttaca 300gttaacgaaa aaaggagatt aaaattgatt atgcctgcta gattctatcc taaccgtacc 360aaatatttgc ccttagataa aggcattaar ccttattatc ctgaacacac agttaatcat 420tacttccaaa ctaggcatta yttacatact ctgtggaagg ctggtatttt atataagaga 480gaaactactc gcagcgcctc attctgtggg tcaccatatt cttgggaaca agagctacag 540catgggaggt tcgtattcca aacctcgaca aggcatgggg acgaatcttt ctgttcccaa 600tcctctggga ttctttcccg atcaccagtt ggacccggca ttcagagcca attcaaacaa 660tccagattgg gacttcaacc ccaacaagga tcaatggcck gcggcacacc aggtaggagt 720gggatccttc gggccagggt tcactccacc acacggcaat cttttggggt ggagccctca 780ggctcagggc atrttgacaa cagtrccagc rgcgcctcct cctgcctcca ccaatcggca 840gtcaggaaga cagcctactc ccatctctcc acctctraga gacagtcatc ctcaggccac 900attccaccaa gctctgctag atcccagagt gaggggccta tacyttcctg ctggtggctc 960cagttccgga acagtraacc ctgttccgac tactgcctct cccatatcgt caatcttctc 1020gaggactggg gaccctgcac cgaagatgga gagcaccaca tcaggattcc taggacccct 1080gctcgtgtta caggcggggt ttttcttgtt gacaagaatc ctcacaatac cacagagtct 1140agactcgtgg tggacttctc tcaattttct agggggagca cccacgtgtc ctggccaaaa 1200tttgcagtcc ccaacctcca atcactcacc aacctcttgt cctccaattt gtcctggtta 1260tcgctggatg tgtctgcggc gttttatcat attcctcttc atcctgctgc tatgcctcay 1320cttcttgttg gttcttctgg actaycaagg tatgttgccc gtttgtcctc tacttccagg 1380aacatcaacy accagcacgg gaccatgcaa gacctgcacg actcctgctc aaggaacctc 1440tatgtttccc tcttgttgct gtacaaaacc ttcggacgga aattgcactt gtattcccat 1500cccatcatct tgggctttcg caagattcct atgggagtgg gcctcagtcc gtttctcatg 1560gctcagtttt ctagtgccat ttgttcagtg gttcgtaggg ctttccccca ctgtttggct 1620ttcagttatg tggatgatgt ggtattgggg gccaagtctg tacaacatct tgaatccctt 1680tttaccgctg ttaccaattt tcttttgtct ttgggtatac atttaaaccc tactaaaact 1740aaacgttggg gctactccct tcacttcatg ggwtatgtaa ttggaagttg gggtacctta 1800ccacaggaac atattgtaca caaaatcaaa caatgttttc ggaaacttcc tataaataga 1860cctattgatt ggaaagtatg tcaacgaatt gtggggcttc taggctttgc cgctcccttt 1920acacaatgtg gttacccagc attaatgcct ttgtatgcat gtatacaagc taaacaggct 1980ttcacttttt cgccaactta caaggccttt ctgtgtaaac aatatctgca cctttacccc 2040gttgctcggc aacggtcagg tctttgccaa gtgtttgctg acgcaacccc cactggttgg 2100ggcttggcca taggccatca gcgcmtgcgt ggaacctttg tggctcctct gccgatccat 2160actgcggaac tcctagcagc ttgttttgct cgcagccggt ctggagcaaa cattatcggc 2220accgacaact ctgttgtcct ctctcggaaa tacacctcct ttccatggct gctaggctgt 2280gctgccaact ggatcctgcg cgggacgtcc tttgtctacg tcccgtcrgc gctgaatccc 2340gcggacgacc cgtctcgggg caggttggga ctctaccgtc cccttcttcg tctgccgttc 2400cggccgacca cggggcgcac ctctctttac gcggtctccc cgtctgtgcc ttctcatctg 2460ccggaccgtg tgcacttcgc ttcacctctg cacgtcgcat ggaaaccacc gtgaacgccy 2520gccaggtctt gcccaaggtc ttacataaga ggactcttgg actctcagca atgtcaacga 2580ccgaccttga ggcatacttc aaagactgtg tatttacaga ctgggaggag ttgggggagg 2640agactaggtt aatgatcttt gtactaggag gctgtaggca taaattggtc tgttcaccag 2700caccatgcaa ctttttcacc tctgcctaat catctcttgt tcatgtccca ctgttcaagc 2760ctccaagctg tgccttgggt ggctttgggg catggacatt gacacctata aagaatttgg 2820agcttctgtg gagttactct cttttttgcc ttctgacttc tttccgaata ttcgtgatct 2880cctcgacacc gcctctgctc tgcatcggga kgccttagag tctcmggaac attgttcmcc 2940tcaccataca gcactaaggc aagctattgt gtgttggggt gagttgatga atctggccac 3000ctgggtggga agtaatttgg aagacccagc atccagggaa ttagtagtaa gctatgtcaa 3060cgttaatatg ggcctaaaaa tcagacaact attgtggttt cacatttcct gtcttacttt 3120tggaagagaa actgttcttg agtatttggt gtcttttgga gtgtggattc gcactcctcc 3180cgcttacaga ccaccaa 319710837PRTHepatitis B Virusmisc_feature(234)..(234)Xaa can be Gly or Cys 10Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Asp 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Asp Trp Gln Thr Pro Ser Phe Pro 65 70 75 80 His Ile His Leu Lys Glu Asp Ile Ile Asp Arg Cys Gln Gln Tyr Val 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro 100 105 110 Ala Arg Phe Tyr Pro Asn Arg Thr Lys Tyr Leu Pro Leu Asp Lys Gly 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Thr Val Asn His Tyr Phe Gln Thr 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 165 170 175 Gln Glu Leu Gln His Gly Arg Phe Val Phe Gln Thr Ser Thr Arg His 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Ser 195 200 205 Pro Val Gly Pro Gly Ile Gln Ser Gln Phe Lys Gln Ser Arg Leu Gly 210 215 220 Leu Gln Pro Gln Gln Gly Ser Met Ala Xaa Gly Thr Pro Gly Arg Ser 225 230 235 240 Gly Ile Leu Arg Ala Arg Val His Ser Thr Thr Arg Gln Ser Phe Gly 245 250 255 Val Glu Pro Ser Gly Ser Gly His Xaa Asp Asn Ser Xaa Ser Xaa Ala 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser His 275 280 285 Leu Ser Thr Ser Xaa Arg Gln Ser Ser Ser Gly His Ile Pro Pro Ser 290 295 300 Ser Ala Arg Ser Gln Ser Glu Gly Pro Ile Xaa Ser Cys Trp Trp Leu 305 310 315 320 Gln Phe Arg Asn Ser Xaa Pro Cys Ser Asp Tyr Cys Leu Ser His Ile 325 330 335 Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Thr Glu Asp Gly Glu His 340 345 350 His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe 355 360 365 Leu Val Asp Lys Asn Pro His Asn Thr Thr Glu Ser Arg Leu Val Val 370 375 380 Asp Phe Ser Gln Phe Ser Arg Gly Ser Thr His Val Ser Trp Pro Lys 385 390 395 400 Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn 405 410 415 Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Ile Pro 420 425 430 Leu His Pro Ala Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu 435 440 445 Xaa Arg Tyr Val Ala Arg Leu Ser Ser Thr Ser Arg Asn Ile Asn Xaa 450 455 460 Gln His Gly Thr Met Gln Asp Leu His Asp Ser Cys Ser Arg Asn Leu 465 470 475 480 Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr Phe Gly Arg Lys Leu His 485 490 495 Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly 500 505 510 Val Gly Leu Ser Pro Phe Leu Met Ala Gln Phe Ser Ser Ala Ile Cys 515 520 525 Ser Val Val Arg Arg Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Val 530 535 540 Asp Asp Val Val Leu Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu 545 550 555 560 Phe Thr Ala Val Thr Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn 565 570 575 Pro Thr Lys Thr Lys Arg Trp Gly Tyr Ser Leu His Phe Met Gly Tyr 580 585 590 Val Ile Gly Ser Trp Gly Thr Leu Pro Gln Glu His Ile Val His Lys 595 600 605 Ile Lys Gln Cys Phe Arg Lys Leu Pro Ile Asn Arg Pro Ile Asp Trp 610 615 620 Lys Val Cys Gln Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe 625 630 635 640 Thr Gln Cys Gly Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln 645 650 655 Ala Lys Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys 660 665 670 Lys Gln Tyr Leu His Leu Tyr Pro Val Ala Arg Gln Arg Ser Gly Leu 675 680 685 Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Ile 690 695 700 Gly His Gln Arg Xaa Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His 705 710 715 720 Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala 725 730 735 Asn Ile Ile Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr 740 745 750 Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly 755 760 765 Thr Ser Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro 770 775 780 Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pro Leu Leu Arg Leu Pro Phe 785 790 795 800 Arg Pro Thr Thr Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro Ser Val 805 810 815 Pro Ser His Leu Pro Asp Arg Val His Phe Ala Ser Pro Leu His Val 820 825 830 Ala Trp Lys Pro Pro 835 11226PRTHepatitis B Virusmisc_feature(92)..(92)Xaa can be Thr or Ile 11Met Glu Ser Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 Pro Gly Gln Asn Leu Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu

Xaa Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 115 120 125 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Phe Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Val Met Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 Leu Asn Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 12984DNAHepatitis B Virus 12tccgcctcct gcctctacca atcgacagtc aggacggcag cctaccccgc tgtctccacc 60tctgagaatc actcatcctc aggccatgca gtggaactcc acaaccttcc accaaactct 120gcaagatccc aragtgagag gcctgkmtct ccctgctggt ggctccagtt caggaacagt 180aaaccctgtt ccgactactg cctctcccat atcgwcaatc ttctcgagga ttggggaccc 240tgcgctgaac atggagaaca tcacatcagg attcctagga cccctgctcg tgttacaggc 300ggggtttttc ttgttgacaa gaatcctcac aataccgcag agtctagact cgtggtggac 360ttctctcaat tttctagggg gaactaccgt gtgtcttggc caaaattcgc agtccccaac 420ctccaatcac tcaccaacct cctgtcctcc aacttgtcct ggttatcgct ggatgtgtct 480gcggcgtttt atcatcttcc tcttcatcct gctgctatgc ctcatcttct tgttggttct 540tctggactat caaggtatgt tgcccgtttg tcctctaatt ccaggatcat caaccaccag 600cacgggaccc tgcagaacct gcacgactcc tgctcaagga acctctatgt atccctcctg 660ttgctgtaca aaaccttcgg atggaaactg cacctgtatt cccatcccat catcctgggc 720tttcggaaaa ttcctatggg agtgggcctc agcccgtttc tcttggctca gtttactagt 780gccatttgtt cagtggttcg tagggctttc ccccattgtt tggctttcag ttatatggat 840gatgtggtat tgggggccaa gtctgtatcg catcttgagt ccctttttac cgctgttacc 900aattttcttt tgtctttggg tatacattta aaccctaaca aaacaaaaar awggggttat 960tctctaaatt tcatgggcta tgtc 98413328PRTHepatitis B Virusmisc_feature(49)..(49)Xaa can be Gly or Val 13Ser Ala Ser Cys Leu Tyr Gln Ser Thr Val Arg Thr Ala Ala Tyr Pro 1 5 10 15 Ala Val Ser Thr Ser Glu Asn His Ser Ser Ser Gly His Ala Val Glu 20 25 30 Leu His Asn Leu Pro Pro Asn Ser Ala Arg Ser Gln Ser Glu Arg Pro 35 40 45 Xaa Ser Pro Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser 50 55 60 Asp Tyr Cys Leu Ser His Ile Xaa Asn Leu Leu Glu Asp Trp Gly Pro 65 70 75 80 Cys Ala Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala 85 90 95 Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr 100 105 110 Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn 115 120 125 Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 130 135 140 Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser 145 150 155 160 Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu 165 170 175 Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser 180 185 190 Asn Ser Arg Ile Ile Asn His Gln His Gly Thr Leu Gln Asn Leu His 195 200 205 Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys 210 215 220 Thr Phe Gly Trp Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly 225 230 235 240 Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala 245 250 255 Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His 260 265 270 Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser 275 280 285 Val Ser His Leu Glu Ser Leu Phe Thr Ala Val Thr Asn Phe Leu Leu 290 295 300 Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Xaa Xaa Gly Tyr 305 310 315 320 Ser Leu Asn Phe Met Gly Tyr Val 325 14308PRTHepatitis B Virusmisc_feature(43)..(43)Xaa can be Lys or Arg 14Pro Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Leu 1 5 10 15 Ser Pro Pro Leu Arg Ile Thr His Pro Gln Ala Met Gln Trp Asn Ser 20 25 30 Thr Thr Phe His Gln Thr Leu Gln Asp Pro Xaa Val Arg Gly Leu Xaa 35 40 45 Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val Asn Pro Val Pro Thr 50 55 60 Thr Ala Ser Pro Ile Ser Xaa Ile Phe Ser Arg Ile Gly Asp Pro Ala 65 70 75 80 Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val 85 90 95 Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln 100 105 110 Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Thr Thr 115 120 125 Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro 130 135 140 Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg 145 150 155 160 Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu 165 170 175 Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile 180 185 190 Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Thr Thr 195 200 205 Pro Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro 210 215 220 Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe 225 230 235 240 Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser 245 250 255 Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Ile Val 260 265 270 Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr 275 280 285 Arg Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu 290 295 300 Trp Val Tyr Ile 305 15580DNAHepatitis B Virus 15ttgtcctggt tatcgctgga tgtgtctgcg gcgttttatc atcttcctct tcatcctgct 60gctatgcctc atcttcttgt tggttcttct ggactatcaa ggtatgttgc ccgtctgtcc 120tctagttccg agatcttcaa ccaccagcgc gggacaatgc agaacctgca cgactactgc 180tcaaggaacc tctatgtatc cctcctgttg ctgtaccaaa ccttcggacg gaaattgcac 240ctgtattccc atcccatcat cctgggcttt cggaaaattc ctatgggagt gggcctcagt 300ccgtttctcc tggctcagtt tgctagtgcc atttgttcag tggttcgtag ggctttcccc 360cactgtttgg ctttcagtta tatggatgat gtggtattgg gggccaagtc tgtacaacat 420cttgagtccc tttttaccgc tgttaccaat tttcttttgt ctttgggtat acatttaaat 480cctaacaaaa ctaaaagatg gggttactct ttaaatttca tgggctatgt cattggatgt 540catgggtcct tgccacaaga tcacatcata cagaaaatca 58016193PRTHepatitis B Virus 16Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro 1 5 10 15 Leu His Pro Ala Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu 20 25 30 Ser Arg Tyr Val Ala Arg Leu Ser Ser Ser Ser Glu Ile Phe Asn His 35 40 45 Gln Arg Gly Thr Met Gln Asn Leu His Asp Tyr Cys Ser Arg Asn Leu 50 55 60 Tyr Val Ser Leu Leu Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His 65 70 75 80 Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly 85 90 95 Val Gly Leu Ser Pro Phe Leu Leu Ala Gln Phe Ala Ser Ala Ile Cys 100 105 110 Ser Val Val Arg Arg Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met 115 120 125 Asp Asp Val Val Leu Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu 130 135 140 Phe Thr Ala Val Thr Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn 145 150 155 160 Pro Asn Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr 165 170 175 Val Ile Gly Cys His Gly Ser Leu Pro Gln Asp His Ile Ile Gln Lys 180 185 190 Ile 17158PRTHepatitis B Virus 17Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu 1 5 10 15 Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr 20 25 30 Gln Gly Met Leu Pro Val Cys Pro Leu Val Pro Arg Ser Ser Thr Thr 35 40 45 Ser Ala Gly Gln Cys Arg Thr Cys Thr Thr Thr Ala Gln Gly Thr Ser 50 55 60 Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr 65 70 75 80 Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu 85 90 95 Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val 100 105 110 Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp 115 120 125 Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile Leu Ser Pro Phe 130 135 140 Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 145 150 155 18965DNAHepatitis B Virus 18ctaccaatcg acagtcaggg aggcagccta ccccgctgtc tccacctttg agaaacactc 60atcctcaggc catgcagtgg aactccacaa ctttccacca aactctacaa gatcccaggg 120tgagaggcct gtatttccct gctggtggct ccagttcagg aacagtaaac cctgttccga 180ctactgcctc tcccatatcg tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg 240agaacatcac atcaggattc ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt 300tgacaaaaat cctcacaata ccgcagagtc tagactcgtg gtggacttct ctcaattttc 360tagggggaac caccgtgtgt cttggccaaa attcgcagtc cccaacctcc aatcactcac 420caacctcctg tcctccgact tgtcctggtt atcgctggat gtgtctgcgg cgttctatca 480tattcctctt catcctgctg ctatgcctca tcttcttgtt ggttcttctg gactatcaag 540gtatgttgcc cgtctgtcct ctaattccag gatcktcaac caccagcgcg ggaccatgca 600gaacctgcac gactactgct caaggaacct ctatgtatcc ctcctgttgt tgtaccaaac 660cttcggacgg aaattgcacc tgtattccca tcccatcatc ctgggctttc ggaaaattcc 720tatgggagtg ggcctcagcc cgtttctcct ggctcagttt actagtgcca tttgttcagt 780ggttcgtagg gctttccccc actgtttggc tttcagttat atggatgatg tggtattggg 840ggccaagtct gttcagcmtc gtgaagccct ttttaccgct gttaccaatt ttcttwtgtc 900tttgggtaya catttaaacc ctaacaaaam tagaagatgg ggttattcct taaatttcat 960gggct 96519321PRTHepatitis B Virusmisc_feature(192)..(192)Xaa can be Val or Phe 19Tyr Gln Ser Thr Val Arg Glu Ala Ala Tyr Pro Ala Val Ser Thr Phe 1 5 10 15 Glu Lys His Ser Ser Ser Gly His Ala Val Glu Leu His Asn Phe Pro 20 25 30 Pro Asn Ser Thr Arg Ser Gln Gly Glu Arg Pro Val Phe Pro Cys Trp 35 40 45 Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Asp Tyr Cys Leu Ser 50 55 60 His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Ala Glu His Gly 65 70 75 80 Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly 85 90 95 Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu 100 105 110 Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn His Arg Val Ser Trp 115 120 125 Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser 130 135 140 Ser Asp Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His 145 150 155 160 Ile Pro Leu His Pro Ala Ala Met Pro His Leu Leu Val Gly Ser Ser 165 170 175 Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser Arg Ile Xaa 180 185 190 Asn His Gln Arg Gly Thr Met Gln Asn Leu His Asp Tyr Cys Ser Arg 195 200 205 Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Gln Thr Phe Gly Arg Lys 210 215 220 Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro 225 230 235 240 Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gln Phe Thr Ser Ala 245 250 255 Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cys Leu Ala Phe Ser 260 265 270 Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Val Gln Xaa Arg Glu 275 280 285 Ala Leu Phe Thr Ala Val Thr Asn Phe Leu Xaa Ser Leu Gly Xaa His 290 295 300 Leu Asn Pro Asn Lys Xaa Arg Arg Trp Gly Tyr Ser Leu Asn Phe Met 305 310 315 320 Gly 20304PRTHepatitis B Virusmisc_feature(286)..(286)Xaa can be Ile or Leu 20Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Leu Ser Pro Pro Leu 1 5 10 15 Arg Asn Thr His Pro Gln Ala Met Gln Trp Asn Ser Thr Thr Phe His 20 25 30 Gln Thr Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly 35 40 45 Gly Ser Ser Ser Gly Thr Val Asn Pro Val Pro Thr Thr Ala Ser Pro 50 55 60 Ile Ser Ser Ile Phe Ser Arg Ile Gly Asp Pro Ala Leu Asn Met Glu 65 70 75 80 Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly 85 90 95 Phe Phe Leu Leu Thr Lys Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser 100 105 110 Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Thr Thr Val Cys Leu Gly 115 120 125 Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro 130 135 140 Pro Thr Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Ser Ile Ile 145 150 155 160 Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu 165 170 175 Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser 180 185 190 Thr Thr Ser Ala Gly Pro Cys Arg Thr Cys Thr Thr Thr Ala Gln Gly 195 200 205 Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn 210 215 220 Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu 225 230 235 240 Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu Val Pro 245 250 255 Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Val 260 265 270 Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Phe Ser Xaa Val Lys 275 280 285 Pro Phe Leu Pro Leu Leu Pro Ile Phe Xaa Cys Leu Trp Val Xaa Ile 290 295 300 21938DNAHepatitis B Virus 21ctccaccacg ttccaccaaa ctcttcaaga tcccagagtc agggccctgt actttcctgc 60tggtggctcc agttcaggaa cagtgagccc tgctcagaat actgtctctg ccatatcgtc 120aatcttatcg aagactgggg accctgtacc gaacatggag aacatcgcat caggactcct 180aggacccctg ctcgtgttac aggcggggtt tttctcgttg acaaaaatcc tcacaatacc 240acagagtcta gactcgtggt ggacttctct caattttcta ggggaaacac ccgtgtgtct 300tggccaaaat tcgcagtccc aaatctccag tcactcacca acctgttgtc ctccaatttg 360tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctctgca tcctgctgct 420atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480aattccagga tcatcaacga ccagcaccgg accatgcaaa acctgcacaa cgcctgctca 540aggaacctct atgttwccct catgttgctg tacaaaacct

acggacggaa actgcacctg 600tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660tttctcatgg ttcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720tgtctggctt tcagttatat ggatgatgtg gttttggggg ccaagtctgt acaacmtctt 780gastcccttt atgccgctgt taccaatttt cttctgtctt tgggtataca tttaaaccct 840gacaaaacaa aaarakgggg atattccctc aacttcatgg gatatgtawt tgggagttgg 900ggcacattgc cacaggaaca tattgtmcaa aaaatcaa 93822312PRTHepatitis B Virusmisc_feature(186)..(186)Xaa can be Thr or Ser 22Leu His His Val Pro Pro Asn Ser Ser Arg Ser Gln Ser Gln Gly Pro 1 5 10 15 Val Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Glu Pro Cys Ser 20 25 30 Glu Tyr Cys Leu Cys His Ile Val Asn Leu Ile Glu Asp Trp Gly Pro 35 40 45 Cys Thr Glu His Gly Glu His Arg Ile Arg Thr Pro Arg Thr Pro Ala 50 55 60 Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr 65 70 75 80 Thr Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn 85 90 95 Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 100 105 110 Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser 115 120 125 Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu 130 135 140 Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser 145 150 155 160 Asn Ser Arg Ile Ile Asn Asp Gln His Arg Thr Met Gln Asn Leu His 165 170 175 Asn Ala Cys Ser Arg Asn Leu Tyr Val Xaa Leu Met Leu Leu Tyr Lys 180 185 190 Thr Tyr Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly 195 200 205 Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Met Val 210 215 220 Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His 225 230 235 240 Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser 245 250 255 Val Gln Xaa Leu Xaa Ser Leu Tyr Ala Ala Val Thr Asn Phe Leu Leu 260 265 270 Ser Leu Gly Ile His Leu Asn Pro Asp Lys Thr Lys Xaa Xaa Gly Tyr 275 280 285 Ser Leu Asn Phe Met Gly Tyr Val Xaa Gly Ser Trp Gly Thr Leu Pro 290 295 300 Gln Glu His Ile Val Gln Lys Ile 305 310 23277PRTHepatitis B Virusmisc_feature(185)..(185)Xaa can be Leu or Phe 23Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg Val Arg Ala Leu 1 5 10 15 Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val Ser Pro Ala Gln 20 25 30 Asn Thr Val Ser Ala Ile Ser Ser Ile Leu Ser Lys Thr Gly Asp Pro 35 40 45 Val Pro Asn Met Glu Asn Ile Ala Ser Gly Leu Leu Gly Pro Leu Leu 50 55 60 Val Leu Gln Ala Gly Phe Phe Ser Leu Thr Lys Ile Leu Thr Ile Pro 65 70 75 80 Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Glu Thr 85 90 95 Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Gln Ile Ser Ser His Ser 100 105 110 Pro Thr Cys Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu 115 120 125 Arg Arg Phe Ile Ile Phe Leu Cys Ile Leu Leu Leu Cys Leu Ile Phe 130 135 140 Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu 145 150 155 160 Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr 165 170 175 Thr Pro Ala Gln Gly Thr Ser Met Xaa Pro Ser Cys Cys Cys Thr Lys 180 185 190 Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala 195 200 205 Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Phe 210 215 220 Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr 225 230 235 240 Val Trp Leu Ser Val Ile Trp Met Met Trp Phe Trp Gly Pro Ser Leu 245 250 255 Tyr Asn Xaa Leu Xaa Pro Phe Met Pro Leu Leu Pro Ile Phe Phe Cys 260 265 270 Leu Trp Val Tyr Ile 275 24762DNAHepatitis B Virus 24cagcgagccc tgctcagaat actgtctctg ccatatcgtc aatcttatcg aagactgggg 60accctgtacc gaacatggag aacatcgcat caggactcct aggacccctg ctcgtgttac 120aggcggggtt tttcttgttg acaaaaatcc tcacaatacc acagagtcta gactcgtggt 180ggacttctct caattttcta ggggggacac ccgtgtgtct tggccaaaat tcgcagtccc 240aaatctccag tcactcacca acttgttgtc ctccaacttg tcctggttat cgctggatgt 300atctgcggcg ttttatcatc ttcctctgca tcctgctgct atgcctcatc ttcttgttgg 360ttcttctgga ctatcaaggt atgttgcccg tatgtcctct aattccagga tcatcaacaa 420ccagcaccgg accatgcaaa acctgcacga ctcctgctca aggaacctct atgtttccct 480catgttgctg tacaaaacct acggacggaa actgcacctg tattcccatc ccatcatctt 540gggctttcgc aaaataccta tgggagtggg cctcagtccg tttctcttgg ytcagtttac 600tagtgccatt tgttcagtgg ttcgtagggc tttcccccac tgtctggctt tcagttatat 660ggatgatgtg gttttggggg ccaagtctgc acaacatctt gagtccctty atgccgctgt 720taccaatttt cttttgtctt tgggtataca tttaacccct ca 76225253PRTHepatitis B Virusmisc_feature(197)..(197)Xaa can be Ala or Val 25Ser Glu Pro Cys Ser Glu Tyr Cys Leu Cys His Ile Val Asn Leu Ile 1 5 10 15 Glu Asp Trp Gly Pro Cys Thr Glu His Gly Glu His Arg Ile Arg Thr 20 25 30 Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys 35 40 45 Asn Pro His Asn Thr Thr Glu Ser Arg Leu Val Val Asp Phe Ser Gln 50 55 60 Phe Ser Arg Gly Asp Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro 65 70 75 80 Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu 85 90 95 Ser Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala 100 105 110 Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val 115 120 125 Ala Arg Met Ser Ser Asn Ser Arg Ile Ile Asn Asn Gln His Arg Thr 130 135 140 Met Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu 145 150 155 160 Met Leu Leu Tyr Lys Thr Tyr Gly Arg Lys Leu His Leu Tyr Ser His 165 170 175 Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser 180 185 190 Pro Phe Leu Leu Xaa Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg 195 200 205 Arg Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val 210 215 220 Leu Gly Ala Lys Ser Ala Gln His Leu Glu Ser Leu Xaa Ala Ala Val 225 230 235 240 Thr Asn Phe Leu Leu Ser Leu Gly Ile His Leu Thr Pro 245 250 26250PRTHepatitis B Virusmisc_feature(197)..(197)Xaa can be Leu or Phe 26Ala Ser Pro Ala Gln Asn Thr Val Ser Ala Ile Ser Ser Ile Leu Ser 1 5 10 15 Lys Thr Gly Asp Pro Val Pro Asn Met Glu Asn Ile Ala Ser Gly Leu 20 25 30 Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys 35 40 45 Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn 50 55 60 Phe Leu Gly Gly Thr Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Gln 65 70 75 80 Ile Ser Ser His Ser Pro Thr Cys Cys Pro Pro Thr Cys Pro Gly Tyr 85 90 95 Arg Trp Met Tyr Leu Arg Arg Phe Ile Ile Phe Leu Cys Ile Leu Leu 100 105 110 Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu 115 120 125 Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro 130 135 140 Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser Met Phe Pro Ser 145 150 155 160 Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile 165 170 175 Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val 180 185 190 Arg Phe Ser Trp Xaa Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val 195 200 205 Gly Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Phe 210 215 220 Trp Gly Pro Ser Leu His Asn Ile Leu Ser Pro Phe Met Pro Leu Leu 225 230 235 240 Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 245 250 27818DNAHepatitis B Virus 27gaggattggg gaccctgcgc tgaatatgga gaacatcaca tcaggattcc taggacccct 60tctcgtgtta caggcggggt ttttcttgtt gacaagaatc ctcacaatac cgcagagtct 120agastcgtgg tggacttctc tcaattttct aggggsaacc accgtgtgtc ttggccaaaa 180ttcgcagtcc ccaacctcca atcactcacc aacctcctgt cctccgactt gacctggtta 240tcgctggatg tgactgcggc attttatcat attcctcttc atcctgctgc tatgcctcat 300cttcttgttg gttcttctgg actatcaagg tatgttgccc gtttgtcctc taattccagg 360atcctcaacc accagcacgg gaacatgccg aacttgcacg actcctgctc aaggaacctc 420tatgtatccc tcctgttgct gtaccaaacc ttcggacgga aattgcacct gtattcccat 480cccatcatcc tgggctttcg gaaaattcct atgggagtgg gcctcagccc gtttctcatg 540gctcagtttg gtagtgccat ttgttcagtg gttcgtaggg ctttccccca ctgtttggct 600ttcatttatg tggatgatrt ggtattgggg gccaagtctg tacagcatct tgagtccctt 660tttaccgctg ttaccaattt tcttttgtct ctgggtatac atttgrwccc tmacaaaaca 720aagagatggg gttactccct aaattttatg ggctatgtca ttggatgtta tgggtccttg 780ccacaagaac acatcataca taaaatcaaa gaatgttt 81828272PRTHepatitis B Virusmisc_feature(42)..(42)Xaa can be Leu or Val 28Glu Asp Trp Gly Pro Cys Ala Glu Tyr Gly Glu His His Ile Arg Ile 1 5 10 15 Pro Arg Thr Pro Ser Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys 20 25 30 Asn Pro His Asn Thr Ala Glu Ser Arg Xaa Val Val Asp Phe Ser Gln 35 40 45 Phe Ser Arg Gly Asn His Arg Val Ser Trp Pro Lys Phe Ala Val Pro 50 55 60 Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asp Leu Thr Trp Leu 65 70 75 80 Ser Leu Asp Val Thr Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala 85 90 95 Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val 100 105 110 Ala Arg Leu Ser Ser Asn Ser Arg Ile Leu Asn His Gln His Gly Asn 115 120 125 Met Pro Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu 130 135 140 Leu Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His 145 150 155 160 Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser 165 170 175 Pro Phe Leu Met Ala Gln Phe Gly Ser Ala Ile Cys Ser Val Val Arg 180 185 190 Arg Ala Phe Pro His Cys Leu Ala Phe Ile Tyr Val Asp Asp Xaa Val 195 200 205 Leu Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val 210 215 220 Thr Asn Phe Leu Leu Ser Leu Gly Ile His Leu Xaa Pro Xaa Lys Thr 225 230 235 240 Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Cys 245 250 255 Tyr Gly Ser Leu Pro Gln Glu His Ile Ile His Lys Ile Lys Glu Cys 260 265 270 29225PRTHepatitis B Virusmisc_feature(33)..(33)Xaa can be Asp or Glu 29Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Xaa Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Xaa Thr Thr Val Cys 35 40 45 Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Thr Pro Gly Tyr Arg Trp Met Leu Arg His Phe Ile Ile 65 70 75 80 Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu 85 90 95 Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser 100 105 110 Thr Thr Ser Thr Gly Thr Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly 115 120 125 Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn 130 135 140 Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu 145 150 155 160 Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Val Val Pro 165 170 175 Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Phe 180 185 190 Met Trp Met Xaa Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Leu Ser 195 200 205 Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 210 215 220 Trp 225 30683DNAHepatitis B Virus 30atcgcagtcc ccaacctcca atcactcacc aacctcctgt cctccaactt gacctggtta 60tcgctggatr tgtctgcggc gttttatcat cttcctcttc atcctgctgc tatgcctcat 120cttcttgttg gttcttctgg actatcaagg tatgttgccc gtttgtcctc taattccagg 180atcmtcaacc accagcacgg gaccatgcag racctgcacg actcctgctc aaggaacctc 240tatgaatccc tcctgttgct gtwccraacc ttcggacgga aattgcacct gtattcccat 300cccatcatcc tgggctttcg gaaaattcct atgggagtgg gcctcagccc gtttctcctg 360gctcarttta ctagtgcyat ttgttcagtg gttcgtaggg ctttccccca ctgtktggct 420ttcagttata trgatgatgt ggtattgggg gccaagtctg tacagcatct tgagkccctt 480twtaccgctg ttaccaattt tcttttgtct ctgggtayac atttaaaccc tcacaaaaca 540aaaagatggg gttacymttt acatttcatg ggctatgtca ttggatgtta tgggtcattg 600ccacaagatc acatcakaca gaaaatcaaa gaatgtttta gaaaacttcc tgttaatagg 660cctattgatt ggaaagtatg tca 68331227PRTHepatitis B Virusmisc_feature(24)..(24)Xaa can be Met or Val 31Ile Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn 1 5 10 15 Leu Thr Trp Leu Ser Leu Asp Xaa Ser Ala Ala Phe Tyr His Leu Pro 20 25 30 Leu His Pro Ala Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu 35 40 45 Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser Arg Ile Xaa Asn His 50 55 60 Gln His Gly Thr Met Gln Xaa Leu His Asp Ser Cys Ser Arg Asn Leu 65 70 75 80 Tyr Glu Ser Leu Leu Leu Leu Xaa Xaa Thr Phe Gly Arg Lys Leu His 85 90 95 Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly 100 105 110 Val Gly Leu Ser Pro Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys 115 120 125 Ser Val Val Arg Arg Ala Phe Pro His Cys Xaa Ala Phe Ser Tyr Xaa 130 135 140 Asp Asp Val Val Leu Gly Ala Lys Ser Val Gln His Leu Glu Xaa Leu 145 150 155 160 Xaa Thr Ala Val Thr Asn Phe Leu Leu Ser Leu Gly Xaa His Leu Asn 165 170 175 Pro His Lys Thr Lys Arg Trp Gly Tyr Xaa Leu His Phe Met Gly Tyr 180 185 190 Val Ile Gly Cys Tyr Gly Ser Leu Pro Gln Asp His Ile Xaa Gln Lys 195 200 205 Ile Lys Glu Cys Phe Arg Lys Leu Pro Val Asn Arg Pro

Ile Asp Trp 210 215 220 Lys Val Cys 225 32173PRTHepatitis B Virusmisc_feature(22)..(22)Xaa can be Ile or Met 32Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr 1 5 10 15 Pro Gly Tyr Arg Trp Xaa Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe 20 25 30 Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln 35 40 45 Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser 50 55 60 Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser Met 65 70 75 80 Asn Pro Ser Cys Cys Cys Xaa Xaa Pro Ser Asp Gly Asn Cys Thr Cys 85 90 95 Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp 100 105 110 Ala Ser Ala Arg Phe Ser Trp Leu Xaa Leu Leu Val Xaa Phe Val Gln 115 120 125 Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met 130 135 140 Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Leu Xaa Pro Phe Xaa 145 150 155 160 Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Xaa Ile 165 170

Claims

1. A biotin- or other ligand-labeled primer which hybridizes to the DNA or mRNA which codes for one or more of the isoleucine (I), aspartic acid (D), or valine (V) at a position corresponding to position 236 of SEQ ID NO: 28, valine (V) at a position corresponding to position 204 of SEQ ID NO: 28, methionine (M) at a position corresponding to position 180 of SEQ ID NO: 28, glycine (G) at a position corresponding to position 184 of SEQ ID NO: 28, or isoleucine (I) at a position corresponding to position 202 of SEQ ID NO: 28.

2. A biotin- or other ligand-labeled primer which hybridizes to the DNA or mRNA which codes for a threonine (T) at a position corresponding to position 85 of SEQ ID NO: 28.

3. A biotin- or other ligand-labeled primer which hybridizes to the DNA or mRNA which codes for a leucine (L) or a valine (V) at a position corresponding to position 42 of SEQ ID NO: 28, a threonine (T) at a position corresponding to position 78 of SEQ ID NO: 28, or an asparagine (N) at a position corresponding to position 128 of SEQ ID NO: 28.

4. A kit comprising a primer of claim 1 and one or more additional primers which hybridizes to the DNA or mRNA which codes for one or more additional mutations in the HBV DNA polymerase gene.

5. A kit comprising a primer of claim 2 and one or more additional primers which hybridizes to the DNA or mRNA which codes for one or more additional mutations in the HBV DNA polymerase gene.

6. A kit comprising a primer of claim 3 and one or more additional primers which hybridizes to the DNA or mRNA which codes for one or more additional mutations in the HBV DNA polymerase gene.